United States Environmental Protection Agency Environmental Research Laboratory Gulf Breeze PL 32561 Research.and Development EPA-600/S3-82-018 June 1982 Project Summary Effects of Soluble Fractions of Drilling Fluids and Hexavalent Chromium on the Development of the Crabs, Rhithropanopeus harr/si/and Callinectes sapidus j* V C. G. Bookhout, R. Monroe, R. Forward, and J. D. Costlow, Jr. This study is part of a series of investi- gations undertaken to determine the effect of the discharge of drilling fluids on marine fauna. The first division of this project was to determine the range of concentrations of the mud aqueous frac- tion (MAP) and the suspended particulate phase (SPP) of a low-density Kgnosul- f onate type mud with f errochrome added (No. 4 mud) which would affect swim- ming behavior, survival and duration of development of the mud crab, Rhithrop- anopeus harrisii, and the blue crab, CaH- nectes sapUus, from the time of hatching until the 1 st crab stage is reached. As far as is known, this is the first investigation on the effect of MAP and SPP, soluble fractions of whole mud, on the complete larval development of crabs. These frac- tions would be found in the upper plume of discharges of an oil well. The percent survival from hatching to megalopa and to 1st crab stage was 90% or over in seawatar control and in concentrations from 5% (5,000 ppm) to 100% (100,000 ppm) MAP and SPP in three replicate series of R. harrisii larvae tested. There was differential survival of Calli- nectes sapidus from hatching to 1 st crab stage in concentrations from 5% to 50% MAP and SPP, but no larvae reached the 1 st crab stage in 100% MAP or SPP. Sta- tistical analyses of the data on survival, mortality and behavior are presented. A second division of the project was to determine the effect of hexavalent chromium, Na2CrO4, on the complete larval development of R. harristt and C. sapidus from the time of patching to 1 st crab stage. The effects of different con- centrations of Na2CrO4 on survival and duration from hatch to megalopa and hatch to 1 st crab stage are given, and the most sensitive stages of larvalaVtvel- opment were determined for each •JPcies. Statistical analyses of the data fiH sur- vival, duration, rate of molting, mortality and behavior are presented. This Project Summary was developed by EPA's Environmental Research Labo- ratory, GuHBreexa, FL, to announce key findings of the research project that is f titty documented in a separate report of the same title {sea Project Report ordering information at back). ------- Part I—Effects of Soluble Fractions of Used Light-Weight Lignosulfonate Type Mud on the Complete Larval Development of Crabs, Rhithropanopeus harrisii and Callinectes sapidus Introduction The Jay Exxon well drilling fluid tested had a density of 9.1 Ib/gal and came from a land-based well in Florida. The samples of No. 4 mud were taken at a depth of 3735.9 m (12,257 feet) and were provided for this investigation by the U.S. Environmental Protection Agency, Environmental Research Labo- ratory, Gulf Breeze, Florida. Although the well was land-based, it was believed that the chemical components and phys- ical characteristics of the drilling fluid were similar to those of offshore wells. Investigators have evaluated the tox- icity of five components of whole mud following the classification originally proposed by Neff eta/. (1980). The mud aqueous fraction (MAF) and suspended paniculate phase (SPP) aretwofractions which have been most intensively inves- tigated in toxicity tests and are two frac- tions tested in this investigation. The 100% MAF contains water soluble and fine particulate fractions of 100,000 ppm mud in water. SPP resembles MAF, but it contains a higher concentration of particulates and a lower concentration of volatiles. These two fractions are found in the upper plume of discharge and remain in the water column longer than other fractions, and, hence, may be the fractions which might be expected to affect larvae of marine organisms as well as plankton. At present, the impact of drilling fluids is incompletely known. Most investiga- tions have been acute toxicity tests. They show that drilling fluids have little or no effect on adult marine organisms, but they reveal that larvae and juvenile invertebrates are sensitive to exposure to drilling fluids. In this investigation, chronic tests were employed because a study covering the entire larval develop- ment of crabs would give a better evalu- ation of possible toxicity of drilling fluids in the field than an acute toxicity study of 96 h. A chronic toxicity test would in- clude all periods of molting when crusta- cean larvae are known to be very sensitive to toxic substances. Furthermore, it is possible to determine which stage or stages in the larval development are par- ticularly sensitive if mortality is recorded at each stage of development. Results and Conclusions The percent survival to megalopa and to 1st crab stage of Rhithropanopeus harrisii was 90% or over in seawater control and in all concentrations of MAF and SPP. There was no consistent reduc- tion in survival in concentrations of MAF and SPP compared to survival in sea- water control. Hence, 100% MAF and 100% SPP light-weight lignosulfonate type mud is non-toxic to developing lar- vae of R. harrisii throughout complete larval development. The average percent survival of three series of Callinectes sapidus reared in seawater control and four concentrations of MAF and SPP is listed in Table 1. There was little difference between C. sapidus survival to megalopa and to 1 st crab in 5% MAF and seawater control, but sur- vival in 5% SPP was less than in control. There was differential survival, however, from 5% MAF and SPP to 100% MAF and SPP. Statistical analysis revealed for C. sapidus zoeal survival to megalopa that there was approximately 4% decrease/ 10% increase in MAF @ 50% CONC., and for survival to 1 st crab, there is ap- proximately 3% decrease/10% increase in MAF @ 50% CONC. For C. sapidus zoeal survival to megalopa and for survival to 1 st crab, there was approximately a 5% decrease in survival for a 10% in- crease in SPP near 50% SPP CONC. From Table 1 it can be observed that there was no significant difference in duration in zoeal development of C. sapi- dus and in hatch to 1 st crab in seawater control and in the concentrations of MAF and SPP employed. The results illustrated in Figure 1 show the effect of MAF levels on the mortality of larvae at each of the seven to eight zoeal stages and a megalopa stage through which C. sapidus passes. The percent mortalities on the graph were obtained from the means of the transformed variable. Mortality of larvae in 5 and 25% MAF was not significantly different from mortality in the control in any of the nine developmental stages, but mortality of larvae reared in 50 and 100% MAF was significantly different from the control in every developmental stage. Although larvae in zoeal stage I were most sensi- tive, larvae in zoeal stage II were also very sensitive, for significant increases in mortality over the previous stage oc- curred in this stage in all media (Figure 1). Mortality of C. sapidus larvae in 5% SPP was not significantly different from mortality in the control in any of the nine developmental stages, but mortality of larvae reared in 50 and 100% SPP was significantly different from the control in every developmental stage (Figure 2). For zoeal stage I, mortality in 25% SPP was significantly different from the con- trol at the 0.05 level. As in the MAF experiment, zoeal stages I and II of C. sapidus were the most sensitive in all media. In general, SPP was more toxic to blue crab larvae than MAF. Blue crab larval behavior is affected by exposure to MAF and SPP with the gen- eral effect being a decline in swimming speed. A significant reduction in speed was only observed in 100% MAF, but it was found in all percentages of SPP tested. Callinectes sapidus larvae could be in the vicinity of drilling operations during development and might be found in the upper turbidity plume, but the chances of many of the larvae remaining in the 3 m highly toxic zone, or even in the 15m intermediate toxic zone, around the dis- charge source long enough to suffer mortality are remote. If by chance a few 1 st or 2nd stage zoeae of C. sapidus in the process of molting happened to be entrained within 15 m of discharge, they might be killed or receive irreversible stress, for these zoeae are extremely sensitive. Larvae in other stages could be affected, but not as quickly. ------- Table 1. Average Percent Survival and Average Duration in Days of Zoeal and Megalopa Development of Three Series (Cs l-lll) of C . sapidus in Seawater Control and Different Concentrations of MAP and SPP of Used Lignosulfonate Type Mud Culture Media Salinity 30°/oo Temp. 25 °C Seawater Control 5% MAP 25% MAP 50% MAF 100% MAF 5% SPP 25% SPP 50% SPP 100% SPP Initial No. of Larvae % Survival to Mean Duration of Development in Days per Series Megalopa 1st Crab Zoea Megalopa Hatch to 1st Crab Csl-50 Csll-50 37.3 28.0 34.9 7.8 42.5 Cslll-50 Csl-50 Csll-50 38.0 31.3 34.8 7.3 41.9 Cslll-50 Csl-50 Csll-50 27.3 23.3 34.8 7.1 42.9 Cslll-50 Csl-50 Csll-50 10.0 8.7 35.9 6.6 42.6 Cslll-50 Csl-50 Csll-50 0.7 - - - - Cslll-50 Csl-50 Csll-50 31.3 26.7 32.4 7.3 41.3 Cslll-50 Csl-50 Csll-50 15.3 12.0 36.2 7.3 43.7 Cslll-50 Csl-50 Csll-50 4.0 4.0 35.8 7.8 43.6 Cslll-50 Csl-50 Csll-50 00-- - Cslll-50 ------- 100 SO 70 60 50 • 40 30 20 10 700%-? WO 90 80 70 K 60 fc I f 50 I * 40 30 20 10 100% 50% 25% / // /// IV V Stage VI VII VIII M Figun2 Figure 1. Effect of MAF of used light-weight lignosulfonate type mud on mortality by stages of development of C. sapidus. a. Significantly different from control (0.05) b. Sionificantly different from control (0.01) *. Significant increase over previous stage (0.05) **. Significant increase over previous stage (O.O1) ~/ ll Hi Iv V W VIIVlJl ~M Stage Effect of SPP of used light- weight lignosulfonate type mud on mortality by stages of development ofC. sapidus. a. Significantly different from control (O.O5) b. Significantly different from control (0.01) *. Significant increase over previous stage (0.05) **.Significant increase over previous stage (0.01) ------- Part II—Effects of Hexavalent Chromium on the Complete Larval Development of Crabs, Rhithropanopeus harrisii and Callinectes sapidus Introduction One of the trace metals in drilling fluids which may have a detrimental environ- mental effect is chromium. The toxicity of chromium to marine organisms varies with valence state, pH and oxidation states. Hexavalent chromium (Cr + 6) is stable in seawater. It often appears as a soluble chromate or dichromate and both are powerful oxidants which can readily penetrate biological membranes and irritate cells (Mertz, 1969). Hexava- lent chromium, as chromic oxide, chro- mate or dichromate, reacts with organic matter in acid solution, leading to the tri- valent form (Cr+3). The trivalent form is associated chiefly with paniculate matter such as clay, which suggests that organic paniculate matter may reduce and bind the hexavalent form in solution. Hexa- valent chromium is much more toxic to organisms than trivalent chromium, in part because hexavalent chromium is water soluble and trivalent chromium has a very low solubility in seawater. Chromium is contributed to drilling fluids chiefly by lignosulfonate, which is added in greater amounts as mud weight is increased. Ferrochrome lignosulfonate, brandname "Q-Broxin," and chrome lig- nosulfonate are common additives to drilling fluids which contribute to Cr enrichment. Initially, these additives contain hexavalent salts, but at tempera- tures between 120 and 175 °C hexava- lent chromium is converted to the trivalent state. The properties of both of these additives can be restored at temperatures between 120 and 175°C by adding moreCr + 6. It has been suggested that after drilling fluids are discharged into the ocean, chromium and associated material are released slowly in soluble form from clay particles into the water. Once freed from clay particles, 3 to 7% Cr + 3 through slow oxidation may revert to Cr + 6. From the above discussion, we con- clude that under certain conditions the possibility exists that both trivalent and hexavalent chromium may be in the vicinity of the discharge source. This investigation was undertaken to determine the concentrations of hexa- valent chromium, Na2CrO4, which are nontoxic, sublethal and acutely toxic to the complete larval development of the mud crab, Rhithropanopeus harrisii, and the blue crab, Callinectes sapidus. Results and Conclusions In experiments on the effect of hexa- valent chromium, Na2Cr04, on the de- velopment of Rhithropanopeus harrisii, it was observed that there were no signifi- cant differences between survival from hatching to megalopa and hatching to 1 st crab in seawater control and 1.1 ppm Na2Cr04, but there was differential survival from 1.1 to 29.1 ppmNa2Cr04 (Table 2). The estimated LC50 for zoeal development from hatching to megalopa was 17.8 ppmNa2Cr04, and from hatch- ing to 1 st crab, the estimated LC50 was 13.7 ppm Na2CrO4. Statistical analysis of the data on R. harrisii duration revealed that there was 0.120 ± 0.021 days increase in duration of zoeal development from hatching to megalopa for each ppm added Na2Cr04, and there was 0.122 ± 0.021 days in- crease in total duration time from hatching to 1 st crab for each ppm added Na2Cr04. From Figure 3, it can be seen that 1 ppm Na2Cr04 is nontoxic to /?. harrisii larvae, for there is no more mortality in this concentration than in seawater control. There is differential larval mor- tality from concentrations of 1.1 ppm to 58.1 ppm Na2CrO4. Concentrations of 7.2 ppm and 14.5 ppm Na2CrO4 are considered sublethal, since more than 10 percent of R. harrisii larvae reached the 1st crab stage. Concentrations of 29, 41, 46 and 58 ppm Na2CrO4 are acutely toxic to R. harrisii larvae, since less than 10 percent reached the 1st crab stage in 29 ppm Na2Cr04 and none reached the 1 st crab stage in 41,46 and 58 ppm Na2Cr04. Rhithropanopeus larval swimming speed was affected by exposure to Na2CrO4. In general, low sublethal concentrations caused an increase in swimming speed and concentrations near those which are acutely toxic caused a decline. In experiments on the effect of hexa- valent chromium, Na2Cr04, on the de- velopment of Callinectes sapidus, it was observed that survival from hatching to 1st crab occurred in 1.1 to 4.7 ppm Na2Cr04 (Table 3). There was better survival in 1.1 ppm Na2CrO4 than in sea- water control, but there was differential survival from 1.1 to 7.2 ppm Na2CrO4. The LC50 for complete zoeal develop- ment of C. sapidus was estimated to be 2.9 ppm Na2CrO4, and the LC50 for de- velopment from hatching to 1st crab stage was estimated to be 1.0 ppm Na2Cr04. Statistical analysis of the data on C. sapidus duration revealed that there was 1.65 ±0.29 days increase in duration of zoeal development from hatching to megalopa for each ppm added Na2CrO4, and that there was 1.31 ±0.29 days in- crease in total duration time from hatch- ing to 1 st crab stage for each ppm added Na2Cr04. From Figure 4, it can be seen that there is significantly less mortality of C. sapidus larvae in 1.1 ppm Na2Cr04 than in seawater control. There is also less lar- val mortality in 2.4 ppm Na2Cr04, but it is not significantly different from the control, and hence it is considered non- toxic. There is differential mortality of C. sapidus larvae from concentrations of 4.7 to 7.2 ppm Na2CrO4, and these con- centrations are considered acutely toxic, since less than 10 percent of C. sapidus larvae reached the 1 st crab stage. Blue crab larvae in zoeal stage III were ex- tremely sensitive to 7.2 ppm Na2CrO4, and larvae in zoeal stages III, IV and V were most sensitive in 4.7 ppm Na2CrO4. For most discharges, the background concentration for chromium has been reported to be reached approximately 100 to 150 meters from the point of dis- charge, depending on the amount and the rate of discharge, as well as the currents. Within this area, entrained crab larvae would undoubtedly absorb Cr + 6 more readily than Cr + 3, if both were present, and bioaccumulate chromium. It is ques- ------- Table 2. Average Perct Control and ir Culture Media Salinity 3O°/oo Temp. 25 °C Seawater Control 1. 12 ppm Na2CrO4 7.17 ppm Na2CrO4 14.52 ppm Na2CrO4 29.09 ppm Na2CrO4 int Survival and Average Duration in Days of Zoeal and Megalopa Development of R. harrisii in Seawatei i Different Concentrations of Hexavalent Chromium, Na2Cr04 Initial No. of Larvae % Survival to Mean Duration of Development in Days per Series Megalopa 1st Crab Rhl-50 Rhll-50 Rhlll-50 95.0 93.7 RhlV-5O RhV-50 RhVI-50 Rhl-50 Rhll-50 Rhlll-50 94. 7 93. 7 RhlV-50 RhV-50 RhVI-50 Rhl-50 Rhll-50 Rhlll-50 83.3 68.7 RhlV-50 RhV-50 RhVI-5O Rhl-50 Rhll-50 Rhlll-50 62.7 47.3 RhlV-50 RhV-50 RhVI-5O Rhl-50 Rhll-50 Rhlll-50 22.0 7.0 RhlV-50 RhV-50 RhVI-50 Zoea Megalopa Hatch to Crab 11.9 6.7 19.0 12.1 6.6 18.8 12.7 6.3 19.0 13.6 6.4 19.8 tionable, however, whether crab larvae would remain in the upper turbidity plume long enough to bioaccumulate enough chromium to kill the larvae or to produce sublethal stress. Hence, it is probable that chromium in drilling fluids is not likely to reduce the population of crab larvae and other planktonic organisms in the area around an oil well, except possibly in the immediate vicinity of the discharge pipe. ------- Table 3. Average Percent Survival and Average Duration in Days Through Zoeal and Megalopa Development of Three Series (Cs l-lll) of Callinectes sapidus Reared in Seawater Control and in Different Concentrations of Hsxavalent Chromium, Na2Cr04 Culture Media Salinity 3O%o Temp. 25 °C Initial No. of Larvae per Series % Survival to Mean Duration of Development in Days Megalopa 1st Crab Zoea Megalopa Hatch to 1st Crab Seawater Control 1. 1 ppm Na2Cr04 Csl-50 Csll-50 Cslll-50 Csl-50 Csll-50 Cslll-50 61.3 80.6 38.0 44.7 33.5 34.5 7.6 7.2 39.6 42.0 2.4 ppm Na2CrO4 4.7 ppm Na2Cr04 7.2 ppm Na2Cr04 Csl-50 Csll-50 Cslll-50 Csl-50 Csll-50 Cslll-50 Csl-50 Csll-50 Cslll-50 65.3 14.0 41.3 7.3 36.1 40.0 7.6 8.0 43.2 45.3 100 90 80} ft 70 o S 60 I 50 30 20 10i 0 1 ppm 100 I II III IV M Stage Figure 3. Effect of Na2CrOt in ppm on mortality of ft. harrisii larvae. a. Significantly different from control (0.051 b. Significantly different from control (0.01) * Significant increase over previous stage (0.05) **. Significant increase over previous stage (0.01) ' II III IV V VI VII VIIIM Stage Figure 4. Effect of Na2CrO4 in ppm on mortality of C. sapidus larvae. a. Significantly different from control (0.05) b. Significantly different from control (0.01) *. Significant increase over previous stage (0.05) **. Significant increase over previous stage (0.01) ------- C. G. Bookhout, Robert Monroe, RichardFoward, andJ. D. Costlow. Jr.. are with Duke University Marine Laboratory. Beaufort. NC28516, Charles McKenney, Jr., is the EPA Project Officer (see below). The complete report, entitled "Effects of Soluble Fractions of Drilling Fluids and Hexavalent Chromium on the Development of the Crabs, Rhithropanopeus harrissi and Callinectes sapidus," (Order No. PB 82-197 203; Cost: $9.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Environmental Research Laboratory U.S. Environmental Protection Agency Gulf Breeze, FL 32561 U.S GOVERNMENT PRINTING OFFICE. 1982—559-092/3417 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 ' ,pS 0000329 AGENCY ------- |