Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-10-18 till 2006-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
This study has been performed in compliance with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MeNigu
- Substance type: Intermediate
- Physical state: solid, white
- Analytical purity: 99.7%
- Lot/batch No.: 90771 54
- Expiration date of the lot/batch: June 01,2007
- Solubility in water: < 15 g/kg at 20 °C
- Stability under test conditions: no data
- Storage condition of test material: At room temperature at about 20 "C, away from direct sunlight

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/l MeNigu

- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from the freshly prepared test
media of all test concentrations and the control at the first treatment period (Day 0), at a treatment period in the second week (Day 12), and at a
treatment period in the last week (Day 16). To determine the maintenance of the test item concentrations during the test medium renewal periods,
duplicate samples were taken from the aged test media of all test concentrations and the control at the end of the three test medium renewal periods
mentioned above (i.e. at Days 2, 14 and 19). The samples were taken from the actual test. Prior to sampling, the contents of all replicates per test
concentration were pooled.

- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on preexperiments
(without GLP) for investigation of the storage stability, the test item was determined to be stable in the test water under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior to the start of the test and prior to each test medium renewal, the test medium of the highest nominal test concentration of 100 mg/L was freshly prepared by dissolving 130 mg of the test item completely in 1300 mL of test water by intensive stirring for 10 minutes and ultrasonification for 5 minutes. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were freshly prepared just before introduction of the daphnids (i.e., start of the test) and just prior to each test medium renewal.
- Eluate: not applicable
- Differential loading: The following nominal concentrations of MeNigu were tested: 1.0, 3.2, 10, 32 and 100 mg/L.
- Controls: A control was tested in parallel (test water without test item)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no observations reported

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna Straus
- Justification for species other than prescribed by test guideline: not applicable
- Source: The study was performed with females of a clone of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred in this testing laboratory in culture medium identical to the medium used for the test
- Age of parental stock (mean and range, SD): The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny (no data on mean age, SD).
- Feeding during test
- Food type: food mixture containing one part of green algae of the species Scenedesmus subspicatus (freshly grown in the testing laboratory) and one part of fish food suspension.
- Amount: The food amounts were based on the measured concentrations of total organic carbon (TOC) in the food suspensions.
- Frequency: The test animals were fed on each working day (Monday through Friday)


ACCLIMATION
- No acclimation necessary, since test animals were bred at the testing laboratory and culture medium identical to the medium used for the test
- Health during acclimation (any mortality observed): The health of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
22 d
Post exposure observation period:
not applicable

Test conditions

Hardness:
2.5 mmol/l (= 250 mg/l as CaCO3)
Test temperature:
19-21 °C
pH:
7.9 ± 0.3
Dissolved oxygen:
test water was aerated until oxygen saturation
Salinity:
no data, however detailed mineral compostion of the test water (reconstituted water) is given in the report
Nominal and measured concentrations:
Nominal test item concentrations: 1.0, 3.2, 10, 32 and 100 mg/l
The measured test item concentrations in these test medium samples were between 97 and 100% of the nominal value at the start and the end of the
test medium renewal periods
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open, however the test vessels were covered with glass plates
- Material, size, headspace, fill volume: Each test animal was kept individually in a 100 mL glass beaker containing 80 mL of test medium.
- Aeration: Before use, the test water was aerated until oxygen saturation.
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through experimental design
- Renewal rate of test solution (frequency/flow rate): In this semi-static test, the test media of all test concentrations and of the control were
renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period. In the end, a total of 8 test medium renewals were performed.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (analytical grade salts and additives were dissolved in purified water to obtain the
nominal concentrations of minerals as stated in the study report
- Total organic carbon: no TOC data of reconstituted water; however the amount of food given to the test animals was based on TOC measurements
on the algal and fish food suspensions
- Particulate matter: no data
- Metals: not applicable
- Pesticides: not applicable
- Chlorine: not applicable
- Alkalinity: 0.9 mmo/lL
- Ca/Mg ratio: 6.6
- Conductivity: no data, however detailed mineral compostion of the test water (reconstituted water) is given in the report
- Salinity: no data, however detailed mineral compostion of the test water (reconstituted water) is given in the report
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH values and dissolved oxygen concentrations in the test media were measured in all test
concentrations and in the control at the beginning and end of each test medium renewal period. The water temperature was measured in one
control beaker at the beginning and at the end of each test medium renewal period. The appearance of the test media was visually recorded at each
of the above mentioned intervals.


OTHER TEST CONDITIONS
- Adjustment of pH: 7.9 ± 0.3
- Photoperiod: 16 hours light and 8 hours darkness, with a 30 minute transition period
- Light intensity: 570-740 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The mortality of adults and the number of offspring were recorded at the start of the test, at the first and second day after treatment and thereafter
three times per week before renewal of test media. Dead animals and offspring were removed at the renewal of the test media. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.
The NOEC and the LOEC of the reproduction rate were statistically evaluated by testing the mean reproduction rates at the test concentrations after
21 and 22 days of exposure for statistically significant differences to the control value by multiple Dunnett-tests after one-way analyses of variance

VEHICLE CONTROL PERFORMED: not applicable


RANGE-FINDING STUDY
- Test concentrations: The test concentrations were based on the results of a range finding test (without GLP).
- Results used to determine the conditions for the definitive study: not reported
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: delay of first brood
Duration:
22 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality of parent animals: In the control and at all test concentrations up to and including 100 mgIL, the survival rate of the test animals at the
end of the test was at least 90% or higher. Thus, the survival rate of Daphnia magna after 21 and 22 days was not significantly (>10%) reduced at
any test concentration up to and including 100 mg/L.
- No. of offspring produced per day per female: 5 ± 0.8. After 22 days, the mean reproduction rate in the control was 110 ± 18 living offspring per
adult (mean ± standard deviation). According to the results of a Dunnett-Test (one-sided smaller, a = 0.05), the mean reproduction rates were not
statistically significantly lower than in the control at any test concentration up to and including 100 mg/L.
- Body length and weight of parent animals: no data
- Type and number of morphological abnormalities: no observations reported
- Type and number of behavioural abnormalities: no observations reported
- Number of males and females (parental): The study was started with 10 daphnids per test concentration and control (all female).
- Time to first brood release: 9 days
- Egg development time: no data
- Brood size: no data
- Time to sexual maturity: no data
- Type and magnitude of biochemical changes: no data
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The NOEC and the LOEC of the reproduction rate were statistically evaluated by testing the mean reproduction rates at the test concentrations after
21 and 22 days of exposure for statistically significant differences to the control value by multiple Dunnett-tests after one-way analyses of variance
(ANOVA).

Any other information on results incl. tables

Table 1: The number of living offspring produced per surviving adult within 22 days of exposure

   Nominal concentration of test item (mg/l)         
  Control   1.0  3.2 10   32 100 
 No. of surviving adults  9  10  10 10   10
 Mean no. of living offspring per adult  110.0  95.3  101.0 109.6  105.4   117.8
 CV % 16.1  18.6  17.9   20.0  21.0 10.1 
 % control 100.0   86.6  91.8 99.6  95.5  107.1 
 STAT  n.s.   n.s.    n.s.     n.s.    n.s.

CV %: coefficient of variation in %

STAT: results of a Dunnett-test with the mean value of living offspring (one-sided smaller, α = 0.05)

n.s.: mean value not significantly lower than in the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Since no effect on survival and reproduction of the test animals was determined at the test concentrations up to and including 100 mg/L, the 22-day NOEC was determined to be at least 100 mg/L. The 22-day NOEC might have been higher than 100 mg/L, but concentrations above 100 mg/L were
not tested in accordance with the test guideline.
The lowest concentration tested with toxic effects (22-day LOEC) was determined to be higher than 100 mg/L.
Executive summary:

The effect of the test item MeNigu on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 22 days following the OECD Guidelines for Testing of Chemicals, No. 211. The nominal concentrations of the test item tested were 1.0, 3.2, 10, 32 and 100 mg/L and a control. The samples taken from the highest test concentration of 100 mg/L were analyzed since this test concentration was determined to be the 22-day NOEC. The test item concentrations measured in these samples were between 97 and 100% of the nominal concentration at the start and the end of the test medium renewal periods. Thus, the test item was stable in the test media over the renewal periods of 48 to 72 hours. Therefore, all reported biological results are related to the nominal concentrations of the test item. It was apparent at the observation of the test animals on Day 21 that some test animals would release their offspring from the brood pouch in the next hours. Therefore, the test was extended for 24 hours until Day 22. After 21 and 22 days of exposure, the survival rate of the parental daphnids was at least 90% in the control and at all test concentrations up to and including 100 mg/L. Furthermore, no effect of the test item on the reproduction of the daphnids was determined up to and including the highest test concentration of 100 mg/L. Since no effect on survival and reproduction of the test animals was determined at any test concentration up to and including 100 mg/L, the 22-day NOEC (highest concentration of MeNigu tested without toxic effects after the exposure period of 22 days) was determined to be at least 100 mg/L. The lowest concentration tested with toxic effects (22-day LOEC) was determined to be higher than 100 mg/L.