Registration Dossier

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:
12 August 2016 - 26 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: white solid
- Storage conditions: in refrigerator (2-8°C) protected from light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48
Volume: 3.2 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis (≤ -15ºC) .

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: A saturated solution (SS) was prepared at a loading rate of 100 mg/L applying three days of magnetic stirring to ensure maximum dissolution of the test item in test medium. The resulting aqueous mixture was filtered through a membrane and the obtained saturated solution was used as the highest test concentration. The pH was adjusted from 5.7 to 6.1 with a 1 M NaOH (Merck, Darmstadt, Germany) solution. Hereafter, lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light and used glassware was wrapped in aluminium foil. All final test solutions were clear and colourless.

- Controls: test medium without test item or other additives (blank-control), \ test medium without test item but with the additive used in the treatment of the stock solutions (solvent-control).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture with a known history. At least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. 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.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started 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. After 7 days of cultivation half of the medium was renewed twice a week.
- Medium other than test medium: yes, M7
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Test temperature:
20°C throughout the test
pH:
7.1 - 8.1 throughout the test
Dissolved oxygen:
8.5 - 9.0 mg O2/L
Nominal and measured concentrations:
A combined limit/range-finding test was conducted. Only the controls and the highest concentrations were analysed.
Nominal test concentrations: 1.0, 10 and 100 mg/L
Measured test concentration: 85 mg/L
Test concentrations remained stable throughout the test
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, open, fill volume: 80 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per intermediate concentration (replicates): 2
- No. of vessels per control and highest concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: RO-water
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: yes, pH of all test solutions was adjusted from 5.7 to 6.1 with a 1 M NaOH (Merck, Darmstadt, Germany) solution
- Photoperiod: 0 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, for the highest concentration and the control. Temperature of the medium: continuously in a temperature control vessel beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (July 2016)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- No immobility was observed at any of the concentrations tested (see table 2 below)
- No biological, behavioural or other abnormalities were observed.
- Measured concentration at t=48 was 95% from initial loading rate

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 48h-EC50: 0.39 mg/L, 95%-CI 0.33-0.44 mg/L
- Other: results fell within the historical range.
Reported statistics and error estimates:
The 24h-EC50 and 48h-EC50 could not be determined because the observed effects were below 50%, i.e. no effects were observed.

Any other information on results incl. tables

Table 1          Concentrations of the test item in test medium

Time of sampling1 [hours]

Percentage of SS2 [%]

Analysed concentration [mg/L]

Relative to initial [%]

 

 

 

 

0

0

n.d.

 

 

100

85.2

 

 

 

 

 

48

0

n.d.

n.a.

 

100

81.0

95

 

 

 

 

1Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

n.d.Not detected.

n.a.Not applicable.

Table 2 Number of introduced daphnids and incidence of immobility in the test

Time (h)

Replicate

Phthaloyl-L-glutamic acid anhydride % SS prep. at 100 mg/L

Control

1.0

10

100

0

A

5

5

5

5

B

5

5

5

5

C

5

5

D

5

5

Total introduced

20

10

10

20

24

A

0 (1)

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

 

 

 

 

48

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1). In the control, < 10% of the daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.
Conclusions:
The 48h-EC50 was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.
Executive summary:

This study was performed to assess the effect of the test substance on the mobility of Daphnia magna after 48 hours. The study was conducted in accordance with OECD Guidline for Testing of Chemicals No. 202 and GLP under static conditions.

In a combined limit/range-finding test, 20 Daphnia magna (less than 24 hours old) were exposed to the test substance for 48 hours in all-glass, open vessels. The exposure was performed in dark.Test solutions were prepared at nominal concentrations of 1.0, 10 and 100 mg/L. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples from the controls and the highest concentrations were taken and analysed to determine the test concentrations. The measured concentration at t=0 for the highest concentration was 85 mg/L and remained stable during the exposure period (95% of initial at t=48).

No immobility was observed at the controls or any of the concentrations. The 48h-EC50 was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.