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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:
(Range-finding study) 5 August 1999 to 7 August 1999 (Definitive study) 5th October 1999 to 7th October 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: March 23rd 1998 Date of signature: July 21st 1998
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/l (nominal concentration).

- Sampling method: Replicates pooled at 0 and 48 hours.

- Sample storage conditions before analysis: Duplicate samples and samples of the 1.8, 5.6, 18 and 56 mg/I test groups were taken and stored frozen (approximately -20ºC) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
The test material was prepared by direct solution in water.
An amount of test material (500 mg) was dissolved in reconstituted water with the aid of ultrasonication and shaking for approximately 20 minutes and the volume adjusted to 5 litres to give the 100 mg/I test solution. Aliquots (20, 36, 64, 112, 200, 360, 640 and 1120 ml) of the 100 mg/l test solution were each separately dispersed in reconstituted water and the volume adjusted to 2 litres to give the remainder of the test series of 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg/l, respectively.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea.
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): Less than 24 hours old (1st instar).
- Method of breeding: Parthenogenesis.
- Feeding during test: (Adult Daphnia) maintained in polypropylene vessels containing approximately 2 litres of reconstituted water. Each culture was fed daily with a suspension of mixed algae (predominantly Chlorella sp.). Gravid adults were isolated the day before initiation of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.
Hardness:
250 mg CaCO3/L.
Test temperature:
21ºC.
pH:
7.9 (at 0-h and 48-h).
Dissolved oxygen:
(0-h) 8.3 mg O2/l,
(48-h) 8.0 - 8.1 mg O2/l.

Salinity:
Not applicable.
Nominal and measured concentrations:
(nominal)
Range-finding test: 0, 0.10, 1.0, 10 and 100 mg/l.
Definitive test: 0, 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.

Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: 300 ml ground glass stoppered conical flasks.
- Aeration: No.
- Renewal rate of test solution: Not renewed.
- No. of organisms per vessel: 10.
- No. of vessels per concentration (replicates): 2.
- No. of vessels per control (replicates): 2.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water.
Stock Solutions
a) CaCl2.2H2O : 11.76 g/l
b) MgSO4.7H2O: 4.93 g/l
c) NaHCO3: 2.59 g/l
d) KCl: 0.23 g/l
Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of < 5 µS cm-1. The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

OTHER TEST CONDITIONS
- Adjustment of pH: pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl (deionised water).
- Photoperiod: The lighting cycle to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/l mg/l (nominal concentration)
- Results used to determine the conditions for the definitive study: No immobilisations up to 10 mg/l. However, immobilisation was observed at 100 mg/l.
Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/I were selected for the definitive study.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%CL of 75 - 91 mg/l
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%CL of 38 - 53 mg/l
Duration:
48 h
Dose descriptor:
other: maximum concentration tested giving no observed effect
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Not reported.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Not reported.

Cumulative immobilisation data in the definitive study

Nominal concentration (mg/l)

Cumulative immobilized Daphnia

(initial population: 10 per replicate)

24 hours

48 hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

0

5.6

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

0

32

0

0

0

0

2

3

5

25

56

0

0

0

0

7

6

13

65

100

8

7

15

75

10

10

20

100

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 45 mg/I with 95% confidence limits of 38 - 53 mg/I. The maximum concentration tested giving no observed effect at 48 hours was 18 mg/l.
Executive summary:

Methods: A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202, "Daphnia sp, Acute immobilisation Test and Reproduction Test" referenced as Method C2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedures: Following a preliminary range-fiding study, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Results: The 48 -hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 45 mg/l with 95% confidence limits of 38 - 53 mg/l. The maximum concentration tested giving no observed effect at 48 hours was 18 mg/l.

Analysis of the test solutions at 0 and 48 hours showed the measured test concentrations to be in the range 80% to 114% of nominal; therefore, the results are based on nominal test concentrations only.

Description of key information

OECD Guideline 202, EU Method C2, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 45 mg/L (95% CL: 38-53 mg/L) based on analytically confirmed nominal test concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
45 mg/L

Additional information

Based on the results of one key study (SPL, 1999), performed according to OECD Guideline 202 and EU Method C2 with GLP statement, the 48 -hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 45 mg/L with 95% confidence limits of 38 - 53 mg/L. The maximum concentration tested giving no observed effect at 48 hours was 18 mg/L.

Analysis of the test solutions at 0 and 48 hours showed the measured test concentrations to be in the range 80% to 114% of nominal; therefore, the results are based on nominal test concentrations.