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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 2005 - 21 January 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study followed Good Laboratory Practice Standards and relevant guidelines with the following notable exception: study deviated from OECD 202 as noted below.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Measured concentrations of the test substance were not within 20% of nominal, and the EC's were reported using nominal concentrations; guidelines state that measured concentrations should be used in such a case to determine the ECs.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
GLP compliance:
yes
Remarks:
with the following exceptions: the characterization of the test substance, and the stability of the test substance under the conditions of storage at the test site, were not determined in accordance with GLP standards.
Analytical monitoring:
yes
Details on sampling:
Samples were collected from one test chamber of each treatment and control group approximately 24 hours prior to the start of the test to confirm the conditioning of the diluter. Samples also were collected from alternating replicate test chambers in each treatment and control group at test initiation and termination to measure concentrations of the test substance. All samples were collected at mid-depth, placed in glass vials, and analyzed as soon as possible without storage.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: One stock solution was prepared for each of the five concentrations tested. A primary stock solution was prepared in DMF at a nominal concentration of 50 mg DBS/mL. Aliquots of the primary stock solution were proportionally diluted with DMF to prepare four secondary stock solutions at concentrations of 3.1, 6.3, 13 and 25 mg/mL. All stocks were mixed by inversion and were clear and light yellow in appearance. The stock solutions were injected into the diluter mixing chambers (at a rate of 15.5 μL/minute) where they were mixed with well water (at a rate of 155 mL/minute) to achieve the desired test concentrations.
- Controls: The solvent control was prepared by injecting DMF into the mixing chamber for the solvent control.
- Chemical name of vehicle: DMF dimethyl formamide
- Concentration of vehicle in test medium: The concentration of DMF in the solvent control and all DBS treatment groups was 0.1 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test solutions appeared clear and colourless in the test chambers and the diluter mixing chambers at test initiation and termination.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: Wildlife International, Ltd., Easton, Maryland.
- Age at study initiation (mean and range, SD): Daphnid neonates used in the test were less than 24 hours old
- Method of breeding: Neonate daphnids were obtained for testing from four individual adult daphnids that had produced at least one previous brood.
- Feeding during test Neonates were not fed during the test.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: During the 14-day holding period immediately preceding the test, water temperatures in the cultures ranged from 19.7 to 20.9ºC, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.8 to 9.0 mg/L (≥87% of saturation), measured with a Yellow Springs Instruments Model 51B dissolved oxygen meter.
- Type of food: Daphnids in the cultures were fed daily a mixture of yeast, Cerophyll® and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum
- Feeding frequency: The adults were fed daily prior to test initiation
- Health during acclimation (any mortality observed): The adult daphnids used to supply neonates for the test were held for at least 13 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding period.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Hardness was measured at test initiation at 132 mg/L as CaCO3 (range 128-136 mg/L).
Test temperature:
Water temperature was measured in each test chamber at the beginning and end of the test, and was within the 20 ± 1 °C range established for the test.
pH:
pH was measured in alternating replicate test chambers of each treatment and control group at the beginning and end of the test and at approximately 24 hours during the test. Measurements of pH were 8.4 throughout the study.
Dissolved oxygen:
Dissolved oxygen was measured in alternating replicate test chambers of each treatment and control group at the beginning and end of the test and at approximately 24 hours during the test. Dissolved oxygen concentrations remained ≥8.7 mg/L (97% of saturation) throughout the test.
Salinity:
NA
Nominal and measured concentrations:
Nominal test concentrations selected were 0.31, 0.63, 1.3, 2.5 and 5.0 mg DBS/L. When the measured concentrations of the test samples collected at 0 and 48 hours were averaged, the mean measured concentrations for the study were 0.15, 0.39, 0.68, 1.3 and 2.7 mg/L, representing 48, 62, 52, 52 and 54% of nominal concentrations, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test compartments were constructed from 300 mL glass beakers that were 6.5 cm in diameter and 12 cm in height and contained approximately 250 mL of test solution. Nylon screen was attached to two holes on the sides of each beaker to allow the test water to flow through the compartment. One test compartment was suspended in each test chamber. The depth of the test water in a representative compartment was approximately 8.7 cm. Test chambers were 25-L Teflon-lined stainless steel aquaria filled with approximately 22 L of test water. The depth of the test water in a representative chamber was approximately 29 cm. Test chambers were indiscriminately positioned in a temperature-controlled water bath set to maintain the desired test temperature. The water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross-contamination.
- Type (delete if not applicable): open / closed
- Aeration: The water was aerated with spray nozzles
- Type of flow-through: proportional diluter
- Renewal rate of test solution : Each test chamber received at least five volume additions of test water every 24 hours. The proportion of test water that was split into each replicate was checked prior to the test to ensure that flow rates varied by no more than ±10% of the mean for the two replicates.
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): Performed in duplicate
- No. of vessels per control (replicates): Performed in duplicate
- No. of vessels per vehicle control (replicates): Performed in duplicate

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm and passed through an ultraviolet sterilizer to remove microorganisms and particles.
- Total organic carbon: <1 mg/L
- Metals (ppm or mg/L): Aluminium <0.204, Arsenic <0.0102, Beryllium <0.0051, Cadmium <0.0051, Calcium 28.2, Chromium <0.0102, Cobalt <5.1, Copper <0.0255, Iron <5.1, Magnesium 11.6, Manganese <0.0153, Mercury <0.0002, Molybdenum <0.0005, Nickel <5.1, Potassium 5.45, Selenium 0.009, Silver <0.0102, Sodium 18.6, Zinc <0.0204
- Pesticides : All pesticides were present at levels below 250 ppb, with the majority at less than 50 ppb.
- Alkalinity: 178 mg/L as CaCO3 (Range 176-180 mg/L)
- Conductivity: 310 μmhos/cm
- Culture medium different from test medium: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test

OTHER TEST CONDITIONS
- Photoperiod: A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity: Light intensity at test initiation, measured using a SPER Scientific Model 840006C light meter, was 204 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made periodically to determine the numbers of dead and immobile organisms. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The number of individuals exhibiting signs of toxicity or abnormal behavior was also noted. Observations were made approximately 5, 24 and 48 hours after test initiation.

TEST CONCENTRATIONS
- Range finding study: Nominal test concentrations were selected in consultation with the Sponsor based on the results of exploratory range finding toxicity tests (not presented in the study)
- Test concentrations: 0.31, 0.63, 1.3, 2.5 and 5.0 mg DBS/L
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and immobility
Remarks on result:
other: 1.3-5.0 (95% CL)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and immobility
Remarks on result:
other: 1.2-1.6 (95% CL)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Duration:
48 h
Dose descriptor:
other: no mortality/immobility concentration
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and immobility
Details on results:
- Behavioural abnormalities: No mortalities or immobility were noted in the 0.31 mg/L treatment group. The single lethargic daphnid observed at test termination was not considered to be treatment-related since it was comparable to the negative control. Percent mortality/immobility in the 0.63, 1.3, 2.5 and 5.0 mg/L treatment groups at test termination was 5, 30, 100 and 100%, respectively. Lethargy was observed among surviving daphnids in those treatment groups at 24 and 48 hours. The no mortality/immobility concentration and the NOEC were both considered to be 0.31 mg/L.
- Mortality of control: No mortalities were observed in the control
- Other adverse effects control: One lethargic daphnia at termination
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Quantitative recoveries of fortified control samples (96 to 101%) indicated that the analytical method utilized was acceptable in recovering DBS from aqueous solutions. Measured concentrations of DBS in the test solution samples were low, but were consistent throughout the test. Possible causes of the low recoveries were low water solubility, adsorption to glass surfaces and particles in the test solutions, and polymerization of the styrene double bond.
Reported statistics and error estimates:
The results of the study were based on the nominal test concentrations. The mortality and immobility data were analyzed using the computer program of C. E. Stephan. The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. In this study, the binomial method was used to calculate the 24-hour EC50 value, and the probit method was used to calculate the 48-hour EC50 value. The no mortality/immobility concentration and the NOEC were determined by visual interpretation of the mortality, immobility and observation data.

The analytical result and percent recovery of test material were calculated using the following:
Concentration of DBS in sample (mg/L) = ((sum of peak areas – y-intercept)/slope) x dilution factor

Percent of nominal concentration = Measured concentration of sample (mg/L)/Nominal concentration of sample (mg/L) x 100

Table 5 (Reproduced from study report)

Cumulative Mortality, Immobility, and Observations

Nominal Test Concentration (mg/L) Replicate Daphnia/Replicate 5 Hours 24 Hours 48 Hours Percent Immobile and Dead
Cumulate Dead Number Immobile Effects1 Cumulate Dead Number Immobile Effects1 Cumulate Dead Number Immobile Effects1
Negative Control A 10 0 0 10 AN 0 0 10 AN 0 0 10 AN 0
  B 10 0 0 10 AN 0 0 10 AN 0 0 1 C; 9 AN  
Solvent Control A 10 0 0 2 Q,AN; 8 AN 0 0 10 AN 0 0 10 AN 0
  B 10 0 0 10 AN 0 0 4 Q,AN; 6 AN 0 0 10 AN  
0.31 A 10 0 0 1 Q,AN; 9 AN 0 0 10 AN 0 0 10 AN 0
  B 10 0 0 10 AN 0 0 10 AN 0 0 1 C; 9 AN  
0.63 A 10 0 0 1 Q,AN; 9 AN 0 0 7 C; 3 AN 0 1 5 C; 4 AN 5
  B 10 0 0 2 Q,AN; 8 AN 0 0 4 C; 6 AN 0 0 7 C; 3 AN  
1.3 A 10 0 0 1 Q,AN; 9 AN 0 0 6 C; 4 AN 0 4 6 C 30
  B 10 0 0 10 AN 0 0 4 C; 6 AN 0 2 7 C; 1 AN  
2.5 A 10 0 0 1 Q,AN; 9 AN 0 1 8 C; 1 AN 6 4 -- 100
  B 10 0 0 3 Q,AN; 7 AN 0 8 2 C 7 3 --  
5.0 A 10 0 0 2 Q,AN; 8 AN 10 0 -- 10 0 -- 100
  B 10 0 0 1 Q,C; 9 AN 10 0 -- 10 0 --  

1 Observed Effects: AN = appear normal; Q,AN = trapped at surface but appear normal after gentle submersion; C = lethargic; Q,C = trapped at surface and appear lethargic after gentle submersion.

Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed for 48 hours under flow-through conditions to five nominal concentrations of dibromostyrene (DBS) ranging from 0.31 to 5.0 mg/L. The test was conducted according to relevant OECD and EPA OPPTS guidelines. Based on the nominal test concentrations, the 48-hour EC50 value was 1.4 mg/L, with a 95% confidence interval of 1.2 to 1.6 mg/L. The no mortality/immobility concentration and the NOEC were 0.31 mg/L.

Description of key information

The cladoceran, Daphnia magna, was exposed for 48 hours under flow-through conditions to five nominal concentrations of dibromostyrene (DBS) ranging from 0.31 to 5.0 mg/L. The test was conducted according to relevant OECD and EPA OPPTS guidelines.  Based on the nominal test concentrations, the 48-hour EC50 value was 1.4 mg/L, with a 95% confidence interval of 1.2 to 1.6 mg/L. The no mortality/immobility concentration and the NOEC were 0.31 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.4 mg/L

Additional information

The key study Palmer SJ et al (2005), was considered adequate for assessment of the short-term toxicity to aquatic invertebrates as a stand alone study. The study was conducted with guideline OECD 202 and in compliance with GLP. The study was assigned a reliability score of 1.