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:
19 February 1997 - 21 February 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
MMBC
Appearance: Light brown solid
Storage conditions: In darkness at room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The test substance rapidly hydrolysed to MMBA.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Nominal test concentrations: 4.58, 9.96, 21.9, 45.8 and 99.6 mg test substance equivalents/L.
Mean measured test concentrations: 4.90, 10.7, 23.2, 46.6 and 100 mg test substance equivalents/L.

Test concentrations were verified by chemical analysis. 10 mL samples were taken from the pooled replicates of control and each test concentration at 0 hours and after 48 hours.

Test solutions

Vehicle:
no
Details on test solutions:
DILUTION WATER
The test organisms were maintained and the tests conducted in a reconstituted medium Elendt M4. The medium was prepared using analytical grade reagents and reverse purified deionised water.

Elendt M4 medium
-Trace elements mg/L

H3BO3 2.85
MnCI2.4H2O 0.36
LiCl 0.31
RbCI 0.071
SrC12.6H2O 0.152
NaBr 0.016
Na2MoO4.2H2O 0.063
CuCl.2H2O 0.0165
ZnCl2 0.013
CoCI2.6H2O 0.010
KI 0.0033
Na2. SeO3 0.0022
NH4VO3 0.00058
Fe-EDTA solution 3.54


-Macro nutrients mg/L

CaCl2.2H2O 294
MgSO4.7H2O 123
KCl 5.80
NaHCO3 64.8
Na2.SiO3.5H2O 6.83
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184


-Vitamins mg/L

Thiamine hydrochloride 0.075
Cyanocobalamine (B 12) 0.0010
Biotine 0.00075

TEST SUBSTANCE PREPARATION
The test substance was added to test water to give an initial stock solution of 99.6 mg test substance equivalents/L and ultrasonicated for approximately 90 minutes so that test substance was hydrolysed to MMBA. The pH of this stock solution was then adjusted using 0.5 mL 1N sodium hydroxide to a pH of 7.9. Serial dilutions of this stock solution were prepared with test water to give the desired series of exposure levels.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Straus
- Source: Cultured in-house and was obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France.
- Age at study initiation (mean and range, SD): The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.
- Method of breeding: Culture conditions ensured that cultures reproduce by parthenogenesis.
- Feeding during test: No.

ACCLIMATION
-Acclimation conditions: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 600 mL of Elendt M4 culture medium with a 16 hour light: 8 hour dark photoperiod at 20 ± 1 °C.
- Type and amount of food: Cultures were fed with a suspension of the unicellular green alga, Chlorella vulgaris.
- Feeding frequency: Daily.

Study design

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

Test conditions

Test temperature:
20 ± 1 °C
pH:
7.2 - 7.9
Dissolved oxygen:
7.9 - 9.3 mgO2/L
Nominal and measured concentrations:
Nominal test concentrations: 4.58, 9.96, 21.9, 45.8 and 99.6 mg test substance equivalents/L.
Mean measured test concentrations: 4.90, 10.7, 23.2, 46.6 and 100 mg test substance equivalents/L.
Details on test conditions:
EXPOSURE CONDITIONS
Five duplicate concentrations were prepared including an untreated control. The test vessels were 250 mL capacity glass jars.

Ten first instar Daphnia were placed without conscious bias into glass jars, each containing 200 mL of prepared test medium or diluent medium to give a loading of 20 mL test solution per organism. The jars were loosely covered to minimise evaporative losses.

Daphnia were exposed to the test or control conditions for a period of 48 hours without renewal of test media.

ENVIRONMENTAL CONDITIONS
Photoperiod of 16 hours light: 8 hours dark and without supplementary aeration or feeding during the 48 hour exposure period.
The temperature in each vessel was measured daily and the pH and dissolved oxygen levels recorded at the start and at the end of the study.

CRITERION OF EFFECT
Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
79.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits (mg MMBC equivalents/L) 44.4 - 326
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
EVALUATION OF DATA
EC50 values were calculated using a logistic model (Berkson, 1944) for which 95% confidence limits were estimated by the likelihood ratio method (Williams, 1986).

Cumulative immobilisation data are given in Table 1. The test substance rapidly hydrolysed to MMBA with a half life of <15 minutes at 25°C in water. Based on this data, the compound in this study was initially converted to the hydrolysis product MMBA in order to test the compound up to a concentration of 99.6 mg test substance equivalents/L. The method of analysis investigated the concentration of MMBA, however, all results are expressed as the mean measured concentration of test substance equivalents (see Table 2). Measured concentrations ranged from 102 to 107% of nominal at 0 hours and 99.1 to 108% of nominal at 48 hours.

Analysis of the immobility data gave the following results:
Highest test concentration resulting in 0% immobilisation: < 4.90* mg test substance equivalents/L
Lowest test concentration resulting in 100% immobilisation: > 100 mg test substance equivalents/L

* 5% Daphnia immobilisation was observed at 48 hours in the control and the lowest test concentration. These levels of immobilisation are not considered to be biologically significant and therefore the no observed effect concentration (NOEC) is 4.90 mg test substance equivalents/L.

Any other information on results incl. tables

Table 1 Cumulative Immobilisation Data for Daphnia magna

Nominal Concentration (mg test substance equivalents/L)

Mean Measured Concentration (mg test substance equivalents/L)

Cumulative Immobilised Daphnia magna

(initial population: 10 per replicate)

 

24 hours

48 hours

R1

R2

Total

%

R1

R2

Total

%

Control

4.58

9.96

21.9

45.8

99.6

Control

4.90

10.7

23.2

46.6

100

0

0

0

0

0

2

0

0

0

0

1

2

0

0

0

0

1

4

0

0

0

0

5

20

0

1

1

2

2

6

1

0

3

4

5

5

1

1

4

6

7

11

5

5

20

30

35

55

R1 Replicate 1

R2 Replicate 2

 

Table 2

Measured Concentrations - Mean Values and Percentages of Nominal

Nominal Concentration (mg test substance equivalents/L)

Number of Samples Analysed

Mean Measured Concentration (mg test substance equivalents/L)

% Nominal

Control

4.58

9.96

2 h9

45.8

99.6

2

2

2

2

2

2

None Detected

4.90

10.7

23.2

46.6

100

-

107

107

106

102

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 48 h EC50 (immobilisation value) for the test substance with Daphnia magna was determined to be 79.1 mg test substance equivalents/L.
Executive summary:

The acute toxicity of the test substance to aquatic invertebrates was investigated in Daphnia Magna in accordance with the standardised guidelines OECD 202 and EU Method C.2. Groups of twenty, first instar Daphnia, less than 24 hours old, were exposed to hydrolysed test substance for 48 hours at nominal concentrations of 4.58, 9.96, 21.9, 45.8 and 99.6 mg test substance equivalents/L dissolved in Elendt M4 medium (mean measured concentrations 4.90, 1037, 23.2, 46.6 and 100 mg test substance equivalents/L). The compound in this study was initially converted to the hydrolysis product MMBA in order to test the compound up to a concentration of 100 mg/L as required in the standard guidelines. The method of analysis investigated the concentration of MMBA, however, all results were expressed as the mean measured concentration of test substance equivalents. The numbers of immobilised daphnids was recorded for each test and control group after 24 and 48 hours. Under the conditions of the study, the 48 hour EC50 was determined to be 79.1 mg test substance equivalents/L.