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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:
From 25 June, 2012 to 7 Sep, 2012
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:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: At t=0 h and t=48 h
Volume: 2 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
DPHA, a UVCB substance, was not completely soluble in test medium at the loading rates initially prepared. Weighing of the test substance was performed under either dimmed or yellow light. Preparation of test solutions was performed under dimmed light conditions.

All test concentrations were prepared separately applying two days of magnetic stirring in the dark to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 3 h where after the Water Accommodated Fractions (WAFs) were siphoned off and used as test concentrations. All WAFs were observed to be clear and colourless, except for the 100 mg/L WAF prepared for both the combined limit/range-finding test and the final test which were observed to be slightly hazy.

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals, and there was no delay in the production of the first brood.
Characteristics: For the test: selection of young daphnids with an age of < 24 h, from parental daphnids of more than two weeks old.
Start of each batch: With newborn daphnids, i.e. less than 3 d old, by placing about 250 of them into 5 L of medium in an all-glass culture vessel.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
Between 19.0 and 20.0°C
pH:
Between 7.6 and 8.0
Dissolved oxygen:
Between 8.8 and 9.1
Nominal and measured concentrations:
Nominal concentration: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.
Measured concentration:The quantitative analysis of the test substance in water was based on the response of the two major components DPPA and DPHA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analysed. The initial concentrations were 7.7, 12, 29, 44 and 90 mg/L, respectively when based on DPPA and 6.0, 8.4, 19, 25 and 35 mg/L, respectively when based on DPHA. These concentrations remained stable during the test period (99-108% of initial for DPPA and 97-105% of initial for DPHA at the end of the test). Given these results, the effect parameters could be based on initial exposure concentrations. Following a worst case scenario DPHA concentrations were used to calculate the effect parameters, i.e. 6.0, 8.4, 19, 25 and 35 mg/L.
Details on test conditions:
Test duration: 48 h
Test type: Static
Test vessels: 100 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 80 mL of test solution
Light: No illumination
Feeding: No feeding
Aeration: No aeration of the test solutions
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 8.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 18 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval 15-21 mg/L
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
ca. 35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: 95% confidence interval between 25 and 47 mg/L
Details on results:
Behavioural abnormalities: No
- Immobility of control: No immobility was observed.
- Other adverse effects control: No
Results with reference substance (positive control):
The 24 h EC50 was 0.50 mg/L with a 95% confidence interval between 0.44 and 0.57 mg/L.
The 48 h EC50 was 0.30 mg/L with a 95% confidence interval between 0.28 and 0.35 mg/L.

Reported statistics and error estimates:
The EC50-value was calculated at 48 h of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Table 1. Acute immobilisation of daphnids after 24 and 48 h in the final test

Loading rate1

DPHA

(mg/L)

Vessel

number

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

Number

Total %

Number

Total %

Control

 

 

 

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

10 (6)

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

18 (8.4)

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

32 (19)

A

B

C

D

5

5

5

5

2

2

1

2

35

4

4

2

4

70

56 (25)

A

B

C

D

5

5

5

5

3

1

1

1

30

4

4

5

3

80

100 (35)

A

B

C

D

5

5

5

5

1

0

0

2

15

5

4

4

4

85

1WAF prepared at the given loading rate

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 48 h EC50 for the test substance was determined to be 18 mg/L (measured initial, WAF).
Executive summary:

A study was conducted to assess the acute toxicity of the test substance, DPHA, to Daphnia magna, according to OECD Guideline 202, EU Method C.2 and ISO standard 634, in compliance with GLP. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to WAFs prepared at loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 48 h and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major components DPHA and DPPA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analyzed. The initial concentrations were 7.7, 12, 29, 44 and 90 mg/L, respectively, when based on DPPA and 6.0, 8.4, 19, 25 and 35 mg/L, respectively, when based on DPHA. These concentrations remained stable during the test period (99-108% of initial for DPPA and 97-105% of initial for DPHA at the end of the test) and did not deviate from the nominal loading rates by more than 20%. Given these results, the effect parameters could be based on initial exposure concentrations or could be based on the nominal loading rates. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test substance did not induce acute immobilisation of Daphnia magna at a loading rate of 18 mg/L after 48 h of exposure (NOELR) (equivalent to 8.4 mg/L of the constituent DPHA (NOEC)). Under the study conditions, the 48 h E(L)C50 was 18 mg/L (equivalent to the nominal loading rate of 35 mg/L) (Bouwman, 2013).

Description of key information

In the short term toxicity study to fish, the initial measured concentrations were 7.7, 12, 29, 44 and 90 mg/L, respectively, when based on DPPA and 6.0, 8.4, 19, 25 and 35 mg/L, respectively, when based on DPHA. These concentrations remained stable during the test period (99-108% of initial for DPPA and 97-105% of initial for DPHA at the end of the test). Overall, the deviation between the nominal loading rates and the measured concentrations is less than 20%. Given these results, the effect parameters could be based on initial measured concentrations or even based on nominal loading rates. In absence of information on the actual moiety driving the toxicity, it was decided to base the effect concentrations on the initial loading rates. Based on nominal loading rate, the 48 h EL50 was 35 mg/L (equivalent to measured concentration of 18 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
18 mg/L

Additional information

A study was conducted to assess the acute toxicity of the test substance, DPHA, to Daphnia magna, according to OECD Guideline 202, EU Method C.2 and ISO standard 634, in compliance with GLP. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to WAFs prepared at loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 48 h and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major components DPHA and DPPA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analyzed. The initial concentrations were 7.7, 12, 29, 44 and 90 mg/L, respectively, when based on DPPA and 6.0, 8.4, 19, 25 and 35 mg/L, respectively, when based on DPHA. These concentrations remained stable during the test period (99-108% of initial for DPPA and 97-105% of initial for DPHA at the end of the test) and did not deviate from the nominal loading rates by more than 20%. Given these results, the effect parameters could be based on initial exposure concentrations or could be based on the nominal loading rates. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test substance did not induce acute immobilisation of Daphnia magna at a loading rate of 18 mg/L after 48 h of exposure (NOELR) (equivalent to 8.4 mg/L of the constituent DPHA (NOEC)). Under the study conditions, the 48 h EC50 was 18 mg/L (equivalent to the nominal loading rate of 35 mg/L) (Bouwman, 2013).