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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
Also followed was the Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
It was not possible to develop a stable analytical method for the specific analysis of the test item. It was consequently decided to analyse the Total Organic Carbon (TOC) concentrations as a surrogate measure.
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 40 mL from the approximate centre of the test vessels
Storage Samples were stored in a refrigerator (2-8°C) until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 40 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batches of Mercaptamine tested were white powders with a purity of 98.8-99.4% and not completely soluble in test medium at the loading rates initially prepared.

For the range-finding test, preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

For the final test, preparation of test solutions started with loading rates individually prepared at 2.2, 4.6, 10, 22, 46 and 100 mg t.i./L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Water Accommodated Fractions were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical 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 hours, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
20 - 21 °C
pH:
7.9 -9.0
Dissolved oxygen:
8.0 - 9.0 mg/l
Nominal and measured concentrations:
WAF loading rate: 2.2, 4.6, 10, 22, 46 and 100 mg/l
Nominal TOC concentrations: 0.69, 1.4, 3.1, 6.9, 14, 31 mg/l
Details on test conditions:
Test Procedure and Conditions
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 50 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 21 minutes after preparation of the test solutions.


Reference substance (positive control):
yes
Remarks:
not concurrent

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
ca. 14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The responses recorded in this test allowed for reliable determination of an EL50. After 48 hours of exposure, all daphnids exposed to the two highest WAFs were immobilized, while 30% and 95% of the daphnids exposed to the WAFs prepared at 10 and 22 mg t.i./L, respectively, were immobilized. No immobility was observed in the control or the two lowest WAFs during the test period.
Results with reference substance (positive control):
the actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the test lab.
The 24h-EC50 was 0.87 mg/L with a 95% confidence interval between 0.80 and 0.95 mg/L.
The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L.

Any other information on results incl. tables

Samples taken from all test groups were analysed. The TOC concentrations measured increased with increasing loading rate, indicating an appropriate preparation of the test solutions. The TOC content of the test item was calculated to be 31.14% based on the provided molecular formula. The initial TOC concentrations in the four highest test concentrations corresponded to 106 -116% of the nominal TOC concentrations as based on the applied loading rates. The TOC concentrations measured in the two lowest test concentrations were at 129 -159% of the nominal TOC concentrations. The measured concentrations were close to the limit of quantification of 1.0 mg TOC/L and may thus not be as reliable as the higher concentrations. At the end of the test, the measured TOC concentrations had remained stable, i.e. were at 105 -112% of the initial concentrations.

Since TOC-analysis is a non-specific method, the effect parameters were determined based on loading rates.

Table 1: Number of Introduced Daphnids and Incidence of Immobility in the Range-Finding Test

Time (h)

Replicate

Mercaptamine;% SS prep. at 100 mg/L

Control

1.0

10

100

0

A

5

5

5

5

B

5

5

5

5

Total introduced

10

10

10

10

24

A

0

0

0

5#

B

0

0

0

5

Total immobilised

0

0

0

10

Effect %

0

0

0

100

 

 

 

 

48

A

0

0

2

5

B

0

0

1

5

Total immobilised

0

0

3

10

Effect %

0

0

30

100

#Microscopic observation revealed no test item attached to the daphnids,

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Mercaptamine; WAF loading rate (mg t.i./L)

 

Control

2.2

4.6

10

22

46

100

0

A

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

 

24

A

0

0

0

0

4

4

5

B

0

0

0

0

3

3

5

C

0

0

0

0

5

3

5

D

0

0

0

1

5#

4

5*

Total immobilised

0

0

0

1

17

14

20

Effect %

0

0

0

5

85

70

100

 

 

 

 

 

 

 

48

A

0

0

0

1

4

5

5

B

0

0

0

2

5

5

5

C

0

0

0

2

5

5

5

D

0

0

0

1

5

5

5

Total immobilised

0

0

0

6

19

20

20

Effect %

0

0

0

30

95

100

100

#Microscopic observation revealed no test item attached to the daphnids.
* Dissolved oxygen: 8.7 mg/L. 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 48h-EL50 for Daphnia magna exposed to Mercaptamine was 14 mg t.i./L based on nominal loading rates (95% confidence interval between 11 and 17 mg t.i./L).