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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
Study period:
2017-03-13 to 2017-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

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

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 and 24 hours) and at the end of both exposure intervals (24 and 48 hours). At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels.- Sample storage conditions before analysis: room temperature

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The stock solution (100 mg/L of the test item was weighed out) was freshly prepared with dilution water prior to the start of the exposure and at renewal (0 and 24 hours). The stock solution was mixed thoroughly by manual agitation.
The highest concentration level of 100 mg/L was tested pH-neutralized in addition to the un-neutralized concentration level to distinguish toxic effects of the test item from pH effects. For this purpose, an appropriate aliquot of the highest concentration level was adjusted to pH 7 ± 0.2 by the addition of 1 M NaOH prior to the start of the exposure (0 hours) and at renewal (24 hours) and tested under the same conditions as the un-neutralized concentration.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): not more than 24 h
- Feeding during test: no

ACCLIMATION
- Acclimation period: not necessary
- Type and amount of food: mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10E006 cells/mL.
- Feeding frequency: 5 times per week
- Health during acclimation (any mortality observed): no mortality observed


Study design

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

Test conditions

Hardness:
160-180 mg/L CaCO3
Test temperature:
18-22 ± 1°C
pH:
4.36 (100 mg test item/l un-neutralized) - 8.79
Dissolved oxygen:
8.67 to 9.16 mg/L
Nominal and measured concentrations:
The measured concentration were within ± 20% of the nominal concentrations which indicates that the test item concentrations were sucessfully maintained. Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: size: 50 mL, fill volume: 20 mL
- Renewal rate of test solution (frequency/flow rate): renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: culture medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the highest concentration was adjusted with 1 M NaOH to a pH of 7± 0.2
- Photoperiod:16/8 h light/dark cycles
- Light intensity: 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The nominal concentration of 100 mg/L of the test item resulted in a pH of 4.67 - 4.94 which caused 100% mortality after 48 h, thus, the pH in the highest concentration used in the main study was adjusted to fit the pH range between 6-9. At the other concentrations no effects were obeserved.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
70.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
un-neutralized
Basis for effect:
mobility
Remarks on result:
other: 95% C.l. 50.0 - 100.0
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
neutralized
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
70.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
un-neutralized
Basis for effect:
mobility
Remarks on result:
other: 95% C.l. 50.0 - 100.0
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
neutralized
Basis for effect:
mobility
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50: 2.05 mg/L(Cl 2.00 - 3.71 mg/L)
Reported statistics and error estimates:
The EC10- and the ECso-values (after 24 and 48 hours) were calculated by linear dose-response regression with the software GraphPad Prism5. The highest concentration level resulting in 0% immobilization and the lowest concentration level causing 100% immobilization were used as 95% confidence limits, since only one partial effect was observed. The ECso-value for the reference item was calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

Any other information on results incl. tables

Table 1: Immobilization Rates after 24 and 48 hours of Exposure

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration

[mg/L]

 

IMMOBILIZATION %

 

 

24 hours

48 hours

 

Re licates

Re licates

1

2

 

3

4

Mean

 1

2

3

4

Mean

100 un-neutralized

100

100

 

100

100

100

100% mortality after 24 hours

100 H-neutralized

0

0

 

0

0

0

0

0

0

0

0

50.0

0

0

 

0

0

0

0

0

0

0

0

25.0

0

0

 

0

0

0

0

0

0

0

0

12.5

0

0

 

0

0

0

0

0

0

0

0

6.25

0

0

 

0

0

0

0

0

0

0

0

Control

0

0

 

0

0

0

0

0

0

0

0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 48-hr-acute toxicity of 2-oxoglutaric acid to Daphnia magna was studied under semi-static conditions according to OECD Guideline 202 (2004).  Daphnids were exposed to control and test chemical at nominal concentrations of 6.25, 12.5, 25.0, 50.0, 100.0 mg a.i./L in culture medium for 48 h.
Since the test item is an acid the pH of the culture medium was measured during the preliminary and the main study. 100 mg/L 2-oxoglutaric acid resulted in an acidification of the medium to a range of 4.36-4.76 which is far below the acceptable pH range, thus the medium of the group with the highest concentration was once adjusted with 1M NaOH to a pH of 7 and a second vessel was left unadjusted in order to detect any pH related effect. The un-neutralized 100 mg/L group exhibited a mortality of 100% after 48h. In the neutralized high dose group no mortality and immobility occurred.
Mortality/immobilization was observed daily. 
The 48-hour EC50 was 70.7 mg a.i./L (95 % confidence limits: 50.0 – 100.0 mg/L) taken into account the results of the un-neutralized high dose group.