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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC Directive 91/414 Annex 1 8.2.5 and The Official Journal of the European Communities Directive 92/69/EEC C.2
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
A 5-mL aliquot was collected from each test vessel at each concentration level, including controls, and transferred to a 4-dram vial. A 5-mL aliquot of CS2 was added to each sample then the samples were shaken on a flat bed shaker for 10 min. After shaking, the samples were set up-right. Once the layers had separated, an aliquot from the CS2 layer was transferred to an auto sampler vial for analysis by GC with a flame ionization detector (GC/FID). Quantitation was achieved using total height of the two DVB isomer peaks.

On Day 0, four additional samples were collected from the 1A (100 mg/L target concentration) and the 6B (7.80 mg/L target concentration) test vessels in order to determine method precision.

The mixing chamber was sampled on each analysis day during a renewal cycle as the solution was mixing. This sample was extracted with CS2 as described above for the test vessel samples.
Vehicle:
no
Details on test solutions:
Six nominal exposure concentrations ranging from 7.8 to 100 mg/L (based on the results from the probe) were used during this study. Definitive test concentrations were set in a geometric series with a factor of ~1.67. The test solutions were prepared by diluting neat test material to the appropriate volumes.

Laboratory water- The laboratory water was Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment for human consumption. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous C02, carbon-filtered, and irradiated.
Test organisms (species):
Daphnia magna
Details on test organisms:
Instars (i.e., daphnids less than 24-h old) from a laboratory-reared culture of Daphnia magna were used. Rearing conditions were: illumination (cool-white fluorescent) 2045±323 lux; 16-h light/8-h dark photoperiod; temperature 20"C. This species is widely accepted and recommended for toxicity testing]. Daphnids were fed an algal diet of Ankistrodesmus convolutus Corda and Nitzschia frustulum Kutzing four times weekly.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Control: 73-74 mg/L CaCO3
High Dose: 32.1 mg/L- 64-68 mg/L CaCO3
Test temperature:
19.8 - 20.7°C (20 ± 1°C)
pH:
7.4 to 7.6
Dissolved oxygen:
8.3 - 8.5 mg/L
Salinity:
no data
Nominal and measured concentrations:
Nominal: 0, 7.80, 13.0, 21.6, 36.0, 60.0 or 100 mg/L
Mean Measured concentrations: 0, 1.07, 2.09, 3.55, 5.83, 11.8, 32.1 mg/L
Details on test conditions:
Delivery System: An intermittent-flow through proportional diluter designed to deliver up to six test concentrations, a vehicle control and a water control was used during this study. The diluter was calibrated so that the target concentration of the test substance in each treatment below the high concentration was approximately 60 percent of that in the next higher treatment level. The diluter operated as follows: A precision dosing system delivered a designated amount (~500 µL) of neat test material from a glass carboy to a mixing chamber (~5 L) where it was mixed with water and then distributed to toxicant cells. The mixing chamber was equipped with a recirculating pump to facilitate dissolution and mixing of the test material. When the diluter cycles, the test substance from each toxicant cell blends with water from its respective water cell and flows into mixing/splitting chambers. Silicone and glass delivery tubes from the splitter cells provided approximately 45 mL of test solution to each of four replicate test vessels. The splitter were randomly positioned on the diluter. The test vessels were positioned under the splitter cells on one tier, side by side, in a water trough. The temperature of the water in the trough was controlled by an electrictemperature controller set to maintain a temperature of 20 ± 1°C. Diluter and laboratory lighting provided a 16-h light/8-h dark transitional photoperiod during testing. The diluter was calibrated prior to the definitive study. During the test, the diluter provided at least 8 volume turnovers in each test vessel during each 24-hour period.

Test Vessels: The test was conducted in 600 mL borosilicate glass beakers with 13 cm long X 7 cm diameter glass inserts. The inserts had 363 mm stainless steel mesh bottoms and open tops. The test vessels with inserts in place held approximately 525 mL of test solution. Each test vessel were labeled with a unique number for identification purposes.

Definitive Study: The definitive study was conducted with 20 instars (two replicates; ten instars/replicate) exposed to each toxicant concentration and controls. Six nominal exposure concentrations ranging from 7.8 to 100 mg/L (based on the results from the probe) were used during this study. Definitive test concentrations were set in a geometric series with a factor of ~1.67. The test solutions were prepared by diluting neat test material to the appropriate volumes. Daphnid instars were impartially added to each test vessel. The dissolved oxygen concentration remained greater than 3 mg/L during the 48-hour exposure period. Daphnids were not fed during the study. The test vessels were not aerated during the conduct of this study. The study began when the organisms were first placed into the solutions. All daphnia were observed daily and mortality (no visible heartbeat or response to gentle prodding) and immobilization were recorded. Immobilization was defined as those animals which were unable to swim within 15 seconds after gentle agitation of the test vessel. Dead daphnids were not removed from the test vessels during the study. Measurement of dissolved oxygen, pH and temperature were taken at the initiationand termination of the test in each replicate in each exposure concentration and control.

Range-Finding Study: A range-finding study with DVB 55 was conducted using 10 instars (two replicates; five instars/replicate). The test nominal concentrations for the range-finding study ranged from 0.8 to 10 mg/L. The delivery of the test material and housing of the daphnids was similar to the method outlined above. The data collected during this probe indicated that the EC50 was >10 mg/L (nominal concentration).
Reference substance (positive control):
no
Duration:
6 h
Dose descriptor:
EC50
Effect conc.:
2.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL: 2.09-3.55 mg/L, EC50 values were calculated using a binomial test, and the 95% confidence limits were calculated using linear interpolation of arcsine transformed data.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL: 2.09-3.55 mg/L, EC50 values were calculated using a binomial test, and the 95 % confidence limits were calculated using linear interpolation of arcsine transformed data.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL: 2.09-3.55 mg/L, EC50 values were calculated using a binomial test, and the 95% confidence limits were calculated using linear interpolation of arcsine transformed data.
Details on results:
The diluter cycled an average of 100 times/day. This resulted in at least 8 volume turnovers in each test vessel per day. The dissolved oxygen levels were greater than 8.3 mg/L over the 48-hour exposure period. Temperature in the test vessels ranged from 19.8 - 20.7C (20 ± 1°C) and the pH ranged from 7.4 to 7.6.

Analvtical Data: Overall average % of target values ranged from 13.7 to 32.1%. None of the analyses on the lab dilution water control samples exhibited peaks eluting at the retention times of the DVB isomer peaks at a concentration exceeding the limit of quantitation of 0.15 mg DVB/L. Results from the analysis of the mixing chamber samples on days 0 and 2 gave analyzed concentrations of 66.7 and 21.9 mg DVB 55/L lab dilution water, respectively. These values corresponded to 66.7 and 21.9% of the target value of 100 mg DVB 55/L.

The analyzed values for both the mixing chamber samples and the dosed test vessels samples were low compared to the target concentrations. This is not unexpected due to the fact that DVB is a volatile compound (vapour pressure = 0.574 mm Hg at 25"C) and the test was run as an open system. Also, prior screening using an air stripping test similar in design to that used by Mackay et al., showed that aeration effectively removed DVB from water. During each renewal cycle the appropriate amount of formulation was added to the mixing chamber (a large open vessel containing approximately 5 L lab dilution water), and this solution was vigorously stirred using a motor and propeller for -4 min. This vigorous stirring worked to aerate the mixing chamber solution, therefore promoting the loss of test material. Although the analyzed values were low compared to target values, the analyzed concentrations in the test vessels at each dose level over the 2-day period were relatively consistent, showing that the system had reached a state of equilibrium. Using the analytical instrument and conditions listed, the two DVB isomer peaks were well resolved from each other, from the two ethylvinylbenzene isomer peaks, and the solvent peak.

Precision: The variability associated with analytical measurement (also referred to as system precision) was determined by making ten injections each from a high and a low analytical standard associated with the day 0 analysis of study ES-3067. This study was run as one in a series of acute daphnid toxicity tests for three DVB formulations, all of which used the exact same analytical instrument and conditions for the quantitation of two DVB isomer peaks. The low standard was prepared at 0.166 mg DVB/L CS2 and the high standard was prepared at 16.5 mg DVB/L CS2. Relative standard deviation values of 3.81 and 4.59% were achieved for the high and low standards, respectively.

The variability associated with the entire sampling and analytical procedure (method precision) was determined by collecting, extracting, and analyzing five identical aliquots from one replicate vessel at both the 100 and 7.78 mg DVB 55/L lab dilution water target dose levels. Percent relative standard deviation values of 3.13 and 2.10% were achieved for the high and low dose levels, respectively.

Linearitv: The GC instrumentation exhibited a linear response for DVB peak height (the total peak height of the two isomer peaks) over a concentration range extending from -0.15 to 65 mg DVB/L in CS2. This corresponds to ~0.31 to 134 mg DVB 55/L laboratory dilution water based on 56.1% DVB in DVB 55 and 86.3% extraction efficiency (day 2 value). The limit of detection, based on 3 x background noise, was calculated to be 0.01 mg DVB/L in CS2, which is equivalent to 0.021 mg DVB 55/L of laboratory dilution water.
Reported statistics and error estimates:
A computer program was used to calculate the EC50 (mortality and immobility) values and corresponding 95% confidence intervals. This program has three statistical methods available: probit analysis, moving average angle analysis, and binomial probability/non-linear interpolation. The probit analysis and moving average methods calculate both the estimated EC50 value and its confidence interval. The binomial method calculates only the confidence interval, while a point estimate of the EC50 is obtained using non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead. The moving average method is less rigorous, statistically, than the probit; and the binomial/nonlinear interpolation method is less rigorous than the moving average. The order of preference used to select the method for reporting the EC50 values is: probit analysis, moving average and binomial/non-linear interpolation. The computer program attempts to process the data using each of the three methods. However, the appropriateness of a given method is determined in the program by the concentration response data, e.g., the number of concentrations resulting in mortality or immobilization between 0 and 100 percent.

Number of daphnids affected

Mean Analyzed

Number Affected

Concentration (mg/L)

Rep.

6h

24h

48h

0

7A

0

0

0

Laboratory water

7B

0

0

0

1.07

6A

0

0

0

 

6B

0

0

0

2.09

5A

0

0

0

 

5B

0

0

3

3.55

4A

10

10

10

 

4B

10

10

10

5.83

3A

10

10

10

 

3B

10

10

10

11.8

2A

10

10

10

 

2B

10

10

10

32.1

1A

10

10

10

 

1B

10

10

10

EC50 (mg/L*)

 

2.72

2.72

2.72

95% CL

 

2.09-3.55

2.09-3.55

2.09-3.55

*EC50 values were calculated using a binomial test, and the 95% confidence limits were calculated using linear interpolation of arcsine transformed data

Validity criteria fulfilled:
yes
Conclusions:
The 6-, 24- and 48-hour EC50 values were 2.72 mg/L (95% CL: 2.09-3.55).
Executive summary:

Divinylbenzene 55 was tested for the acute toxicity to the daphnid, Daphnia magna Straus according to OECD Guideline 202. The objective of this test was to determine the median effective concentration for immobilization (EC50) of divinylbenzene to the daphnid, Daphnia magna Straus. The study was designed as a 48-hour flow-through test with analyzed dose levels. Duplicate groups of 10 daphnids were exposed to mean analyzed treatment levels of 0 (Laboratory water control), 1.07, 2.09, 3.55, 5.83, 11.8 and 32.1 mg/L.

 

Based on the results of this study, the 6-, 24- and 48-hour EC50 values were 2.72 mg/L with a 95% confidence limits of 2.09-3.55.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC Directive 91/414 Annex I 8.2.5 and The Official Journal of the European Communities Directive 92/69/EEC C.2.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
A 5-mL aliquot was collected from each replicate (A and B) test vessel at each concentration level, including controls, and transferred to a 4-dram vial. A 5-mL aliquot of CS2 was added to each sample then the samples were shaken on a flatbed shaker for 10 min. After shaking, and once the layers had separated, an aliquot from the CS2 layer was transferred to an auto sampler vial for analysis by GC/FID. Quantitation was achieved using total height of the two DVB isomer peaks.

On Day 0, four additional samples were collected from the 1A (16.7 mg DVB 63/L target concentration) and the 6B (1.30 mg DVB 63/L target concentration) test vessels in order to determine method precision.

The mixing chamber was sampled each analysis day during a renewal cycle as the solution was mixing. This sample was extracted with CS2 as described above for the test vessel samples.
Vehicle:
no
Details on test solutions:
Six nominal exposure concentrations ranging from 0.148 to 2.64 mg/L (based on the results from the probe) were used during this study. Definitive test concentrations were set in a geometric series with a factor of ~1.67. The test solutions were prepared by diluting neat test material to the appropriate volumes.
Test organisms (species):
Daphnia magna
Details on test organisms:
Instars (i.e. daphnids less than 24-h old) from a laboratory-reared culture of Daphnia magna were used. Rearing conditions are: illumination (cool-white fluorescent) 2045 ± 323 lux; 16-h light/8-h dark photoperiod; temperature 20 ± 2°C. Ths species is widely accepted and recommended for toxicity testing. Daphnids were fed an algal diet of Ankistrodesmus convoltittis Corda and Nitzschia frzistzilurn Kiitzing four times weekly.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Control: 69 mg/L CaCO3
High dose (2.64 mg/L): 67 mg/L CaCO3
Test temperature:
19.0-20.3°C
pH:
7.4-7.6
Dissolved oxygen:
8.1-8.4 mg/L (>92% of saturation)
Salinity:
None
Nominal and measured concentrations:
Nominal: 0, 1.30, 2.16, 3.60, 6.00, 10.0 or 16.7 mg/L
Measured: 0, 0.148, 0.274, 0.465, 0.797, 1.23 or 2.64 mg/L
Details on test conditions:
Test Substance Delivery Svstem: An intermittent-flow through proportional diluter designed to deliver up to six test concentrations, a vehicle control and a water control was used during this study. The diluter was calibrated so that the target concentration of the test substance in each treatment below the high concentration was approximately 60 percent of that in the next higher treatment level. The diluter operated as follows: A precision dosing system delivered a designated amount (~90 µL) of neat test material from a glass carboy to a mixing chamber (~5 L) where it was mixed with water and then distributed to toxicant cells. The mixing chamber was equipped with a recirculating pump to facilitate dissolution and mixing of the test material. When the diluter cycled, the test substance from each toxicant cell blended with water from its respective water cell and flows into mixing/splitting chambers. Silicone and glass delivery tubes from the splitter cells provided approximately 40 mL of test solution to each of four replicate test vessels. The splitter cells were randomly positioned on the diluter. The test vessels were positioned under the splitter cells on one tier, side by side, in a water trough. The temperature of the water in the trough was controlled by an electric temperature controller set to maintain a temperature of 20 ± 1°C. Diluter and laboratory lighting provided a 16-h light/8-h dark transitional photoperiod during testing. The diluter was calibrated prior to the definitive study. During the test, the diluter was set to provide at least 6 volume turnovers in each test vessel during each 24 hour period.

Test Vessels: The test was conducted in 600 mL borosilicate glass beakers with 13 cm long X 7 cm diameter glass inserts. The inserts had 363 mm stainless steel mesh bottoms and open tops. The test vessels with inserts in place held approximately 525 mL of test solution. Each test vessel was labeled with a unique number for identification purposes.

Definitive Study: The definitive study was conducted with 20 instars (two replicates; 10 instars/replicate) exposed to each toxicant concentration and controls. Six nominal exposure concentrations ranging from 0.148 to 2.64 mg/L (based on the results from the probe) were used during this study. Definitive test concentrations were set in a geometric series with a factor of ~1.67. The test solutions were prepared by diluting neat test material to the appropriate volumes. Daphnid instars were impartially added to each test vessel. The dissolved oxygen concentration was greater than 3 mg/L during the 48-hour exposure period. Daphnids were not fed during the study. The test vessels were not aerated during the conduct of this study. The study began when the organisms were first placed into the solutions.

Range-Finding Study: A range-finding study with DVB 63 was conducted using 10 instars (2 replicates; 5 instars/replicate). The test concentrations for the range-finding study ranged from 0.8 to 10 mg/L nominal. The delivery of the test material and housing of the daphnids was similar to the method outlined above. The data collected during this probe indicated that the EC50 was between 6 and 10 mg/L.

Data Recording: All daphnia were observed daily and mortality (no visible heartbeat or response to gentle prodding) and immobilization was recorded. Immobilization is defined as those animals which are not able to swim within 15 seconds after gentie agitation of the test vessel. Dead daphnids were not removed from the test vessels during the study. Measurements of dissolved oxygen, pH and temperature were taken at the initiation and termination of the test in each replicate in each exposure concentration and control.
Reference substance (positive control):
no
Duration:
6 h
Dose descriptor:
EC50
Effect conc.:
> 2.64 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL- >2.64 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 2.64 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL- >2.64 mg/L, EC50 and 95% CI determined by moving average method
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CL: 1.23 - 2.64 mg/L, EC50 and 95% CI determined by moving average method
Details on results:
The diluter cycled an average of 96 times/day. This resulted in at least 7 volume turnovers in each test vessel per day. The dissolved oxygen levels were greater than 92% of saturation (8.1 - 8.4 mg/L) over the 48-hour exposure period. Temperature in the test vessels ranged from 19.0 - 20.3°C (20 ± 1°C) and the pH ranged from 7.4 to 7.6.

Analytical Data: Overall average percent of target values ranged from 11.4 to 15.8%. Results from the analysis of the mixing chamber samples on days 0 and 2 gave analyzed concentrations of 1.69 and 3.34 mg DVB/L lab dilution water, respectively. These values corresponded to 10.1 and 20.0% of the target value of 16.7 mg DVB/L. The analyzed values for both the mixing chamber samples and the dosed test vessels samples were low compared to the target concentrations. This is not unexpected due to the fact that DVB is a volatile compound (vapor pressure = 0.574 mm HG at 25°C) and the test is run as an open system. Also, prior screening using an air stripping test similar in design to that used by Mackay et al., showed that aeration effectively removed DVB from water. During each renewal cycle the appropriate amount of formulation was added to the mixing chamber (a large open vessel containing approximately 5 L lab dilution water), and this solution was vigorously stirred using a motor and propellor for ~4 min. This vigorous stirring, necessary to get homogeneity, worked to aerate the mixing chamber solution, therefore increasing the loss of test material. Although the analyzed values were low compared to target values, the analyzed concentrations in the test vessels at each dose level over the 2-day period were relatively consistent, showing that the system had reached a state of equilibrium. Using the analytical instrument and conditions listed, the two DVB isomer peaks were well resolved from each other, from the two ethylvinyl benzene isomer peaks, and from the solvent peak.

Precision: The variability associated with analytical measurement (also referred to as system precision) was determined by making ten injections each from a high and a low analytical standard associated with the Day 0 analysis. The low standard was prepared at 0.166 mg DVB/L CS2 and the high standard was prepared at 16.5 mg DVB/L CS2. Relative standard deviation values of 3.81 and 4.59% were achieved for the high and low standards, respectively. The variability associated with the entire sampling and analytical procedure (method precision) was determined by collecting, extracting, and analyzing five identical aliquots from one replicate vessel at both the 16.7 and 1.30 mg DVB 63/L lab dilution water target dose levels. Percent relative standard deviation values of 3.13 and 8.72% were achieved for the high and low dose levels, respectively.

Linearity: The GC instrumentation exhibited a linear response for DVB peak height (the total peak height of the two isomer peaks) over a concentration range extending from ~0.05 to 16.5 mg DVB/L CS2 using the conditions listed. This corresponds to ~0.093 to 30.8 mg DVB 63/L laboratory dilution water based on 63.5% DVB in DVB 63 and 84.5% extraction efficiency (day 0 value). A limit of detection, based on 3x background noise, was calculated to be 0.02 mg DVB/L CS2, which is equivalent to 0.037 mg DVB 63/L laboratory dilution water.
Reported statistics and error estimates:
A computer program was used to calculate the EC50 (mortality and immobility) values and corresponding 95% confidence intervals. This program has three statistical methods available: probit analysis, moving average angle analysis, and binomial probability/non-linear interpolation. The probit analysis and moving average methods calculate both the estimated EC50 value and its confidence interval. The binomial method calculates only the confidence interval, while a point estimate of the EC50 is obtained using non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead. The moving average method is less rigorous, statistically, than the probit; and the binomial/non-linear interpolation method is less rigorous than the moving average. The order of preference used to select the method for reporting the EC50 values is: probit analysis, moving average and binomial/non-linear interpolation. The computer program attempts to process the data using each of the three methods. However, the appropriateness of a given method is determined in the program by the concentration-response data, e.g., the number of concentrations resulting in mortality or immobilization between 0 and 100 percent.

Summary of Survival Data

Mean Analyzed

Number Affected

Concentration (mg/L)

Rep.

6h

24h

48h

0 (water control)

7A

0

0

0

 

7B

0

0

0

0.148

6A

0

0

0

 

6B

0

0

1

0.274

5A

0

0

0

 

5B

0

1

1

0.465

4A

0

0

4

 

4B

0

0

2

0.0797

3A

0

0

4

 

3B

0

0

3

1.23

2A

0

1

4

 

2B

0

0

5

2.64

1A

0

1

10

 

1B

0

2

9

Number affected includes the number dead plus the number showing sublethal effects (immobility).

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 concentration was 1.31 mg DVB-63/L with 95% confidence intervals of 1.23-2.64. Mortality and immobility were noted at concentrations through 0.148 mg DVB 63/L.
Executive summary:

Divinylbenzene 63 was tested for acute toxicity to the daphnid, Daphnia magna Straus in accordance with OECD Guideline 202. The objectives of the study were to determine the median effective concentration (EC50) causing immobilization or death of the daphnid, Daphnia magna Straus, by divinylbenzene 63 (DVB 63). The study was designed as a 48-hour flow-through test with analyzed dose levels. Duplicate groups of 10 daphnids were exposed to mean analyzed treatment levels of 0 (standard dilution water control), 0.148, 0.274, 0.465,0.797, 1.23, and 2.64 mg/L.

 

The 48-hour EC50 concentration was 1.31 mg DVB 63/L with 95% confidence intervals of 1.23-2.64. Mortality and immobility were noted at concentrations through 0.148 mg DVB 63/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC Directive 91/414 Annex I 8.2.5 and The Official Journal of the European Communities Directive 92/69/EEC C.2
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
A 5-mL aliquot was collected from each replicate (A and B) test vessel at each concentration level, including controls, and transferred to a 4-dram vial. A 5-mL aliquot of CS2 was added to each sample then the samples were shaken on a flatbed shaker for 10 min. After shaking, and once the layers had separated, an aliquot from the CS2 layer was transferred to an auto sampler vial for analysis by GC/FID. Quantitation was achieved using total height of the two DVB isomer peaks.

On Day 2, four additional samples were collected from the 1A (10 mg DVB HP/L target concentration) and the 6B (0.78 mg DVB HP/L target concentration) test vessels in order to determine method precision.The mixing chamber was sampled each analysis day during a renewal cycle as the solution was mixing. This sample was extracted with CS2 as described above for the test vessel samples.
Vehicle:
no
Details on test solutions:
An intermittent-flow through proportional diluter designed to deliver up to six test concentrations, a vehicle control and a water control was used during this study. The diluter was calibrated so that the target concentration of the test substance in each treatment below the high concentration was approximately 60 percent of that in the next higher treatment level. The diluter operated as follows: A precision dosing system delivered a designated amount (~50 µL) of neat test material from a glass carboy to a mixing chamber (~5 L) where it was mixed with water and then distributed to toxicant cells. The mixing chamber was equipped with a recirculating pump to facilitate dissolution and mixing of the test material. When the diluter cycled, the test substance from each toxicant cell blended with water from its respective water cell and flowed into mixing/splitting chambers. Silicone and glass delivery tubes from the splitter cells provided approximately 40 mL of test solution to each of four replicate test vessels. The splitter cells were randomly positioned on the diluter. The test vessels were positioned under the splitter cells on one tier, side by side, in a water trough. The temperature of the water in the trough was controlled by an electric temperature controller set to maintain a temperature of 20 ± 1°C. Diluter and laboratory lighting provided a 16-h light/8-h dark transitional photoperiod during testing. The diluter was calibrated prior to the definitive study. During the test, the diluter was set to provide at least 6 volume turnovers in each test vessel during each 24 hour period.
Test organisms (species):
Daphnia magna
Details on test organisms:
Instars (i.e. daphnids less than 24-h old) from a laboratory-reared culture of Daphnia magna were used.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Control: 79-83 mg/L CaCO3
High dose (1.18 ppm): 76-79 mg/L CaCO3
Test temperature:
19.0-20.1°C (20 ± 1°C)
pH:
7.5-7.7
Dissolved oxygen:
8.3-8.5 mg/L (>92% saturation)
Salinity:
None
Conductivity:
Control: 220-280 µmho/cm
High dose (1.18 ppm): 210-220 µmho/cm
Nominal and measured concentrations:
Nominal: 0, 0.780, 1.30, 2.16, 3.60, 6.00 and 10.0 mg/L
Measured: 0, 0.084, 0.135, 0.287, 0.498, 0.612 and 1.18 mg/L
Details on test conditions:
At least 20 instars (two replicates; 10 instars/replicate) were exposed to each toxicant concentration and controls. Six analyzed exposure concentrations ranging from 0.0843 to 1.18 ppm (based on the results from the probe) were used during this study. Definitive test concentrations were set in a geometric series with a factor of ~1.67. The test solutions were prepared by diluting neat test material to the appropriate volumes. Daphnid instars were impartially added to each test vessel. The dissolved oxygen concentration should be greater than 3 mg/L during the 48-hour exposure period. Daphnids were not fed during the study. The test vessels were not aerated during the conduct of this study. The study began when the organisms are first placed into the solutions.

Test Vessels: The test was conducted in 600 mL borosilicate glass beakers with 13 cm long X 7 cm diameter glass inserts. The inserts have 363 mm stainless steel mesh bottoms and open tops. The test vessels with inserts in place hold approximately 525 mL of test solution. Each test vessel was labeled with a unique number for identification purposes.

Range-Finding Study: A range-finding study with DVB HP was conducted using 10 instars (2 replicates; 10 instars/replicate). The test concentrations for the rangefinding study ranged from 0.8 to 6.0 mg/L nominal. The delivery of the test material and housing of the daphnids was similar to the method outlined above. The data collected during this probe indicated that the EC50 was between 6 and 10 mg/L.

Data Recording: All daphnia were observed daily and mortality (no visible heartbeat or response to gentle prodding) and immobilization were recorded. Immobilization was defined as those animals which were not able to swim within 15 seconds after gentle agitation of the test vessel. Dead daphnids were not to be removed from the test vessels during the study. Measurement of dissolved oxygen, pH and temperature were taken at the initiation and termination of the test in each replicate in each exposure concentration and control.
Reference substance (positive control):
no
Duration:
6 h
Dose descriptor:
EC50
Effect conc.:
> 1.18 other: ppm
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobilization
Remarks on result:
other: 95% CI >1.18 ppm, EC50 and 95% CI determined by binomial method
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1.18 other: ppm
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CI >1.18 ppm, EC50 and 95% CI determined by binomial method
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.69 other: ppm
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality/immobility
Remarks on result:
other: 95% CI 0.61-0.82 ppm, EC50 determined and 95% CI determined by probit method.
Details on results:
Analytical Data: Overall average % of target values ranged from 10.2 to 13.8%. Results from the analysis of the mixing chamber samples on days 0 and 2 gave analyzed concentrations of 2.76 and 1.78 mg DVB HP/L lab dilution water, respectively. These values corresponded to 27.6 and 17.8% of the target value of 10.0 mg DVB HP/L. The analyzed values for both the mixing chamber samples and the test vessels samples were low compared to the target concentrations. This is not unexpected due to the fact that DVB is a volatile compound (vapour pressure = 0.574 mm HG at 25°C) and the test was run as an open system. Also, prior screening using an air stripping test similar in design to that used by Mackay, et al., showed that aeration effectively removed DVB from water. During each renewal cycle the appropriate amount of formulation was added to the mixing chamber (a large open vessel containing approximately 5 L lab dilution water), and this solution was vigorously stirred using a motor and propeller for ~4 min. This vigorous stirring, necessary to achieve a homogeneity, worked to aerate the mixing chamber solution, therefore promoting the loss of test material. Although the analyzed values were low compared to target values, the analyzed concentrations in the test vessels at each dose level over the 2-day period were relatively consistent, showing that the system had reached a state of equilibrium. Using the analytical instrument and conditions listed, the two DVB isomer peaks were well resolved from each other, from the two ethylvinylbenzene isomer peaks, and from the solvent peak.

Precision: The variability associated with analytical measurement (also referred to as system precision) was determined by making ten injections each from a high and a low analytical standard associated with the Day 0 analysis of study ES-3067. This study was run as one in a series of acute daphnid toxicity tests for three DVB formulations, all of which used the exact same analytical instrument and conditions for the quantitation of two DVB isomer peaks. The low standard was prepared at 0.166 mg DVB/L CS2 and the high standard was prepared at 16.5 mg DVB/L CS2. Relative standard deviation values of 3.81 and 4.59% were achieved for the high and low standards, respectively. The variability associated with the entire sampling and analytical procedure (method precision) was determined by collecting, extracting, and analyzing five identical aliquots from one replicate vessel at both the 10.0 and 0.78 mg DVB HP/L lab dilution water target dose levels. Percent relative standard deviation values of 1.31 and 7.73% were achieved for the high and low dose levels, respectively. The GC instrumentation exhibited a linear response for DVB peak height (the total peak height of the two isomer peaks) over a concentration range extending from ~0.05 to 24 mg DVB/L CS2 using the conditions listed. This corresponds to ~0.08 to 36 mg DVB HP/L laboratory dilution water based on 80.3% DVB in DVB HP and 82% extraction efficiency (day 0 value). A limit of detection, based on 3x background noise, was calculated to be 0.015 mg DVB/L CS2, which is equivalent to 0.023 mg DVB HP/L laboratory dilution water. None of the analyses on the lab dilution water control samples exhibited peaks eluting at the retention times of the DVB isomer peaks at a concentration exceeding the limit of quantitation of 0.05 mg DVB/L CS2 (approximate concentration of the lowest standard analyzed), which is equivalent to 0.076 mg DVB HP/L lab dilution water based on 80.3% DVB in DVB HP and 82.0% extraction efficiency (day 0 value).
Reported statistics and error estimates:
A computer program was used to calculate the EC50 (mortality and immobility) values and corresponding 95% confidence intervals. This program has three statistical methods available: probit analysis, moving average angle analysis, and binomial probability/non-linear interpolation. The probit analysis and moving average methods calculate both the estimated EC50 value and its confidence interval. The binomial method calculates only the confidence interval, while a point estimate of the EC50 is obtained using non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead. The moving average method is less rigorous, statistically, than the probit; and the binomial/non-linear interpolation method is less rigorous than the moving average. The order of preference used to select the method for reporting the EC50 values is: probit analysis, moving average and binomial/nonlinear interpolation. The computer program attempts to process the data using each of the three methods. However, the appropriateness of a given method is determined in the program by the concentration-response data, e.g., the number of concentrations resulting in mortality or immobilization between 0 and 100 percent.

Summary of Survival Data

Mean Analyzed

Number Affected

Concentration (mg/L)

Rep.

6h

24h

48h

0 (water control)

7A

0

0

0

 

7B

0

0

0

0.0843

6A

0

0

0

 

6B

0

0

1

0.135

5A

0

0

0

 

5B

0

1

0

0.287

4A

0

0

0

 

4B

0

0

0

0.498

3A

0

0

1

 

3B

1

1

2

0.612

2A

1

2

6

 

2B

0

0

3

1.18

1A

0

0

8

 

1B

0

2

10

Number affected includes the number dead plus the number showing sublethal effects (immobility).

Validity criteria fulfilled:
yes
Conclusions:
The 48 hour EC50 for DVB-HP is 0.69 ppm (95% CI 0.61-0.82 ppm).
Executive summary:

Divinylbenzene HP was tested for the acute toxicity to the daphnid, Daphnia magna Straus in accordance with OECD Guideline 202. The objective of this test is to determine the median effective concentration for immobilization (EC50) of divinylbenzene HP to the daphnid, Daphnia magna Straus. The study was designed as a 48-hour flow-through test with analyzed dose levels. Duplicate groups of 10 daphnids were exposed to mean analyzed treatment levels of 0 (standard dilution water control), 0.084, 0.135,0.287, 0.498, 0.612, and 1.18 ppm. Based on the results of this study the 48-hour EC 50 was 0.69 mg/L with 95% confidence limits of 0.61-0.82 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The tested concentrations were measured at 0 hour and 24 hours (before exchange of test solution)
Vehicle:
yes
Details on test solutions:
Vehicle/Solvent and Concentrations: 2-Methoxyethanol and HCO-40 were used as solvents in the tests. The concentrations of 2-Methoxyethanol and HCO-40 in stock solution were 200 mg/L each. The total final concentrations of the two solvents for the tests were 20 mg/L.

Stock Solutions Preparations and Stability: The test substance (100 mg) was dissolved in 2-Methoxyethanol (100 mg), and HCO-40 (100 mg) was added. The amount of the test solution was adjusted to 500 mL by dilution water (test substance 200 mg/L)
Test organisms (species):
Daphnia magna
Details on test organisms:
Test Organisms:
a) Age: < 24 hours old
b) Supplier/Source: National Institute for Environmental Studies (JAPAN)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Dilution water: 63 mg/L as CaCO3
Test temperature:
19.9-20.1°C
pH:
7.4-7.9
Dissolved oxygen:
8.1-8.6 mg/L
Salinity:
No data
Nominal and measured concentrations:
Nominal Concentrations (as mg/L): 0, 1.00, 1.20, 1.60, 2.20, 3.00, 5.60 and 10.00
Measured Concentrations (as mg/L): 0.874, 1.08, 1.31,1.92, 2.56, 4.54 and 8.13
Details on test conditions:
Test Conditions:
a) Dilution Water Source: Dechlorinated tap water
b) Dilution Water Chemistry: Hardness (as CaCO3) 63 mg/L, pH 8.1 (22°C), Alkalinity 45 mg/L, Chlorine concentration < 0.01 mg/L
c) Exposure Vessel Type: 100 mL test solution in a 100 mL Glass Beaker covered with Teflon Sheet
d) Nominal Concentrations (as mg/L): 0, 1.00, 1.20, 1.60, 2.20, 3.00, 5.60 and 10.00
e) Vehicle/Solvent and Concentrations: 2-Methoxyethanol and HCO-40 were used as solvents in the tests. The concentrations of 2-Methoxyethanol and HCO-40 in stock solution were 200 mg/L each. The total final concentrations of the two solvents for the tests were 20 mg/L.
f) Stock Solutions Preparations and Stability: The test substance (100 mg) was dissolved in 2-Methoxyethanol (100 mg), and HCO-40 (100 mg) was added. The amount of the test solution was adjusted to 500 mL by dilution water (test substance 200 mg/L).
g) Number of Replicates: 4
h) Individuals per Replicates: 5
i) Water Temperature: 19.9 - 20.1°C
j) Light Condition: 16:8 hours, light-darkness cycle, not more than 1200 lux
k) Feeding: No
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobility
Remarks on result:
other: Based on the measured concentrations, because some data of the measured concentrations were <80% of the nominal concentrations.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobility
Remarks on result:
other: Based on the measured concentrations, because some data of the measured concentrations were <80% of the nominal concentrations.
Details on results:
- Cumulative Number of Dead Parental Daphnia: 48 hours; Control (0), 1.00 mg/L (0), 1.20 mg/L (0), 1.60 mg/L (0), 2.20 mg/L (11), 3.00 mg/L (20), 5.60 mg/L (20) and 10.0 mg/L (20)
- Calculation of toxic values: Based on the measured concentrations, because some data of the measured concentrations were <80% of the nominal concentrations.
Reported statistics and error estimates:
Data Analysis: Binominal method for EC50
Method of Calculating Mean Measured Concentrations (i.e. arithmetic mean, geometric mean, etc.): Geometric Mean
Validity criteria fulfilled:
yes
Conclusions:
The EC50 is 1.87 mg/L and the NOEC is 1.31 mg/L based on the measured concentrations (geometric mean).
Executive summary:

This study was conducted in accordance with OECD Guideline 202. The EC50 is 1.87 mg/L and the NOEC is 1.31 mg/L based on the measured concentrations (geometric mean).

Description of key information

Four GLP-studies according to OECD guideline 202 are available for different grades of the reaction mass of divinylbenzene and ethylstyrene. The grade with the highest toxicity to Daphnia magna was DVB-HP. Two studies were conducted with this grade and the 48-hour EC50s from these studies were 0.69 mg/L and 1.87 mg/L. The geometric mean of these two data points was 1.14 mg/L. This was the lowest toxicity value for aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.14 mg/L

Additional information

Four high quality acute toxicity tests (Klimisch rating 1) with different grades of the reaction mass of divinylbenzene and ethylstyrene were performed with Daphnia magna. One test was performed with DVB-55 (approximately 55% divinylbenzene and 45% ethylstyrene) resulting in a 48-hour EC50 of 2.72 mg/L. Another acute toxicity test with daphnids was performed with DVB-63 (approximately 63% divinylbenzene and 37% ethystyrene) resulting in a 48-hour EC50 of 1.31 mg/L. Two acute toxicity tests with Daphnia magna were performed with DVB-HP (approximately 80% divinylbenzene and 20% ethylstyrene) with reported 48-hour EC50 values of 0.69 mg/L and 1.87 mg/L. Since the EC50 results for DVB-HP were within an order of magnitude, and were based on the same endpoint and species, a geometric mean of 1.14 mg/L was calculated from these two data points. The EC50 of 1.14 mg/L was selected as the key parameter for acute toxicity to aquatic invertebrates since it was the lowest acute toxicity value for this endpoint.