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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April - 21 June 2005
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study with scientifically acceptable deviations.
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
: see below
Principles of method if other than guideline:
- use of Dimethylformamide as solubility aid, although the water solubility of styrene is sufficient to produce a saturated solution to be diluted for preparing the series of required test concentrations.
- Parent animals were not mantained individually
- number of animals used in the control and solvent control studies not clear.
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: At the preparation, the solutions sampled from the container for preparation was used for the initial analysis. Before renewal, the test solutions in one vessel of four test vessels in each level were sampled centrifuged (3,000 rpm for 10 minutes) to remove feed
and used for analysis.
- Sample storage conditions before analysis: no specification
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Desired amount of the test sample was weighed and dissolved in DMF to prepare the stock solution of 50000 mg/L. The stock solution was diluted in DMF to prepare the stock solution of 10000 times of each level. Desired amount of the stock solution (or solvent) was added to dilution water with stirring in a container for preparation of the test solution. Each test solution was prepared for the respective exposure levels, and the prepared test solutions were divided into each test vessel.
- Controls: dilution water control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethylformide,
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 ml/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no undissolved material observed.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain/clone: Clone A
- Source: originally from the University of Sheffield (Sheffield S10 2UQ, United Kingdom)
- Age of parental stock (mean and range, SD): less than 24-hour old
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: 0.1 - 0.2 mg C (organic carbon content)/day/per animal
- Frequency: once daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The number of the offspring was counted and its conditions were observed by the eye observation. After counting and observation, the offsprings were disposed.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
Post exposure observation period:
no post exposure
35,5 - 41.5 mg/L (CaCO3)
Test temperature:
20 ± 1°C
7.5 - 8.0
Dissolved oxygen:
8.5 - 8.9 mg O2/L
no data
Nominal and measured concentrations:
Nominal concentrations: 0.313, 0.625, 1.25, 2.50 and 5.00 mg/L
Geometric mean of the measured concentratiions: 0.249, 0.523, 1.01, 2.06 and 3.84 mg/L.
The recovery of test item in the test solutions were between 71.4 to 103% at preparation and between 64.5 to 92.4% before renewal compared to nominal concentrations.
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): Glass vessel, closed
- Material, size, headspace, fill volume: 730mL test solution per vessel
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): once daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: no data

- Source/preparation of dilution water: Dechlorinated tap water, with sufficient aeration and controlled-temperature
- Total organic carbon: 0.3 mg/L
- Particulate matter: < 1 mg/L
- Metals: < 0.01 mg/L
- Pesticides: < 0.0005 mg/L
- Chlorine: < 0.01 mg/L
- Alkalinity: 36 mg/L
- Ca/mg ratio: 3.93
- Conductivity: 15.8 mS/m

- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: 19.8 µE/m2s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and vitality of parent were regularly observed. The dead animals were removed from the test vessel); The number of eggs, resting or abortion eegs and discrimnation of sex were determined regularly. The number and vitaliy of offspring were counted everyday. After counting and observation, the offsprings were disposed; The quality (pH, oxygen content, temperature and hardness), as well as the concentration of the test solution were determinaed six tine at period of preparation and before renewal once a week, triple repetitions. The light intensity was measured during the exposure.


- Test concentrations: 1.00, 2.00 and 4.00 mg/L, dilution water and solvent controls were tested parallelly.
- Results used to determine the conditions for the definitive study: 90% of suvival rate and 34% of reproduction inhition at the highest concentration (4.00 mg/L).
Reference substance (positive control):
potassium dichromate
21 d
Dose descriptor:
Effect conc.:
1.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
2.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Key result
21 d
Dose descriptor:
Effect conc.:
1.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 1.74-2.04 mg/L
Key result
21 d
Dose descriptor:
Effect conc.:
> 3.84 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
relative concentration of test substance in the solution to the setting value as of preparation: 71.4-103%, before renewal: 64.5-92.4%
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: A 48-hour acute immobilization test, EC50 (48h) = 0.200 mg/L
Reported statistics and error estimates:
Moving average:
The Values were rounded off in accordance with JIS Z 8401 rule B. (JIS ; Japanese Industrial Standards).

1) Mortality of parent animal: Cumulative mortality rate of parent animal which was 0% and 5% respectively in control and solvent control at the end of exposure. There was no statistical significant difference of mortality of parent animal between all test levels and solvent control.

2) Primiparous day: With 10.3 days, the averaging primiparous days of parent animals at the concentration of 3.84 mg/L was significanlly delated then which in the control, solvent control and the other exposure levels (7.0 - 8.0 days).

3) Average number of cumulative live offspring: The average number of cumulative live offspring per a parent animal which was

108 and 106 animals respectively in control and solvent control in 21 days and that of each test level (0.249, 0.523, 1.01, 2.06, and 3.84 mg/L) were 107, 108, 92.6, 60.4, and 3.3 animals, respectively.

4) Size and condition of parent animal: No significant symptom was observe in controls and at the concentrations of 0,249 and 1.01 mg/L.14 days later, transparency of the body color of the test animal was observed at the concentration of 0.523 mg/L and 6 days later, reductions of activity and growth inhibition at the concentration of 2.06 mg/L level, thereafter transparency of body color, immobilization and lethargic state were observed. 3 days later, growth inhibition of the parent animals was observed at the concentration of 3.84 mg/L, thereafter they were obviously smaller than control. Additionally, after the start of exposure, reduction of activity (1 day later), transparency of body color (6 days later) and immobilization (10 days later) were continuously observed during the exposure.

5) Occurrence of resting egg: During exposure, resting eggs did not occur in all of the level. No statistically significant difference on the the number of dead offspring and abortion eggs observed between controls and test levels.

Validity criteria fulfilled:
Executive summary:

Styrene was testet for possible toxicological effects in an chronic test on the water flea Daphnia magna . The LC50 after 21d was found to be 1.88 mg/L (reproduction) and the LC50 after 21d was >3.84 mg/L.

Description of key information

Styrene was testet for possible toxicological effects in an chronic test on Daphnia magna. The NOEC after 21 d was found to be 1.01 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
1.01 mg/L

Additional information

Following the OECD Guideline 211 (Daphnia magna Reproduction Test) the toxicity of styrene to Daphnia magna was determined in a semi-static test conducted according to Good Laboratoty Practice. Test concentration were: 0.313, 0.625, 1,25, 2,50 and 5.00 mg/L. Under the conditions of the study, the EC50 value for reproduction was 1.88 mg/L (1.74 - 2.04 mg/L) and the 21d LC50 for mortality was > 3.84 mg/L. The 21d NOEC and LOEC were determined to be 1.01 mg/L and 2.06 mg/L, respectively.