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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state / Appearance: Solid / red
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. Each test solution was prepared separately (differential loading) by directly adding test substance to test medium according to the loading rate described in the table below and stirring for about 2 days. Undissolved test substance was visible at each loading rate. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). The aqueous fraction was inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect). In the two highest test solutions the Tyndall effect was positive. Then the aqueous fractions were filtered through a membrane filter (pore width 0.2 µm, Whatman OE66). The first 50 mL of filtered solution was discarded (used to condition the filter). The Tyndall effect was negative in the filtered solutions.
Fresh test solutions were prepared and renewed daily to maximize the exposure to all soluble components of the test substance.
The aqueous phase of the tested concentrations were visibly colorless-clear, only the highest test concentration was sligthly pink and clear over the exposure period.
Since the test substance is a multi-component mixture (UVCB), the test solution after separation of the undissolved material is considered a water accommodated fraction (WAF). According to OECD 23, the term “loading rate” should be used to express exposure to a WAF and is considered analogous to the nominal concentration.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978.
From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Age at start of exposure: <24 hours (at least 3rd brood progeny)
- Feeding during test: None
- Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 parthenogenetically reproducing females for a maximum of 4 weeks and fed live unicellular algae (Desmodesmus subspicatus), cultured continuously at the test facility. All individuals in each mass culture originate from a single female and are thus genetic clones. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start of the exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.

ACCLIMATION
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, and temperature (20 ±1°C).

Study design

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

Test conditions

Hardness:
2.20 – 3.20 mmol/L
Test temperature:
19.7 – 20.6 °C (continuous monitoring)
pH:
7.5 – 8.5
Dissolved oxygen:
Must remain >= 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 hours prior to use.
Conductivity:
550 - 650 µS/cm
Nominal and measured concentrations:
0 (control), 0.46, 1, 2.2, 10, 22 and 46 mg/L as loading rate based on test substance mass without a correction for purity or composition
Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL)
- Test volume: 50 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal period was 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.1 animals/mL

TEST MEDIUM / WATER PARAMETERS
A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 202.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 128-645 lux at a wave length of 400-750 nm

EXPERIMENTAL PROCEDURE
The exposure was started by impartially distributing 20 neonate (< 24 hours old) Daphnia magna evenly among the 4 test vessels per test group. The neonate daphnids all originated from the same mass culture and were thus genetic clones (see chapter 3.2). The Daphnia were transferred into the test vessels with minimal culture water by capturing each daphnid in a pipette then gently expelling it onto a Teflon mesh (70 µm) held by forceps. Each daphnid was then immediately transferred from the mesh into the corresponding test vessel from lowest to highest concentration.
Each test vessel was visually checked for immobilized daphnids after 0, 24 and 48 hours. In addition any abnormal behavior or appearance was documented. For test renewal, daphnids in each replicate were captured with a pipette and transferred into test vessels with fresh test solution.
The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, TOC and conductivity) were determined after aeration and prior to use in the test.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily in old and new test solutions.
The pH and dissolved oxygen content of the test solutions was measured after 0h and 24h (new test solutions) and after 24 h and 48 h (old test solutions) in replicate 1 of each tested concentration.
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.
The TOC (Total organic carbon) values of the test concentrations and the control were analyzed at the start and the end of one renewal interval as an additional water quality parameter.

RANGE-FINDING STUDY
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤2.2 should be used, preferably encompassing the range from no effect to 100% immobilization.
In a preliminary test (experimental conduct in accordance with GLP, without a GLP status) the 48 hour EC50 was between 10 and 100 mg/L.
The raw data of the range finding test are archived together with the raw data of this study.
Reference substance (positive control):
yes
Remarks:
sodium chloride

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.871 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
mobility
Details on results:
DISSOLUTION BEHAVIOR
All of the test solutions were visibly clear and colorless after preparation and remained so after 24 and 48 hours of exposure in the test. No undissolved test substance was visible and there were no other remarkable observations.

VALIDITY CRITERIA
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• ≤10% immobilisation in the control
• O2 concentration ≥3 mg/L in control and test vessels

Results with reference substance (positive control):
In order to verify that the Daphnia magna culture is responding normally to toxic stress, tests with a reference substance, sodium chloride, are conducted monthly. Reference substance tests are conducted according to OECD 202 guidelines and in accordance with GLP, but without a GLP status.
The EC50(48h) of the reference substance sodium chloride was 4.18 g/L (experiment date: 09 May 2017, project number: 50E0789/12E060).
This result is within the range of 3.88 – 7.22 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Any other information on results incl. tables

Table 2: Culture medium


pH value

Conductivity
[µS/cm]

Total hardness
[mmol/L]

Acid capacity up to pH 4.3 [mmol/L]

TOC[mg/L]

Date of measurement

8.0

578

2.41

0.85

1.0

07 Jun 2017

 

Table 3: pH and dissolved oxygen content

pH- and oxygen values in the test solutions after 0 h and 24 h (interval 1)

 

Test Group
[mg/L]

pH value
at 0 h

pH value
after 24 h

O2value [mg/L]
at 0 h

O2value [mg/L]
after 24 h

0 (control)

8.0

8.0

8.6

8.8

0.46

8.0

8.0

8.6

8.9

1

8.0

8.0

8.8

8.7

2.2

8.0

8.0

8.8

8.9

4.6

8.0

8.0

8.8

8.7

10

8.0

8.0

8.9

8.9

22

8.0

8.0

8.7

8.8

measured in the replicate 1 of each concentration

 

Table 4: pH- and oxygen values in the test solutions after 24 h and 48 h (interval 2)

 

Test Group
 [mg/L]

pH value
at 24 h

pH value
after 48 h

O2value [mg/L]
at 24 h

O2value [mg/L]
after 48 h

0 (control)

8.0

8.0

8.5

8.9

0.46

8.0

8.0

8.7

9.0

1

8.0

8.0

8.8

9.0

2.2

8.0

8.0

8.7

9.0

4.6

8.0

8.0

8.8

9.0

10

-

-

-

-

22

-

-

-

-

measured in the replicate 1 of each concentration

-not measured, 100% immobilisation after 24 h

Table 5: Total organic carbon (TOC) content

TOC values in the test solutions after 0 h and 24 h in interval 1

 

Loading rate
[mg/L]

after 0 h
[mg/L]

fresh test solution

after 24 h
[mg/L]

aged test solution

0 (control)

1.1

1.2

0.46

2.2

1.2

1

1.7

1.0

2.2

2.0

1.0

4.6

1.3

0.9

10

1.2

1.0

22

1.0

1.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item is very toxic (acute effect) to aquatic invertebrates.