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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2009-03-09 to 2009-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 211 with GLP certificate. All validity criteria were fulfilled and no deviations were observed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected from 23 to 25 August 2006 / Signed on 21 March 2007
Specific details on test material used for the study:
- Water solubility: 100 µg/L
- Vapour pressure: 0.04 Pa at 25°C
- Partition coefficient (log Pow): 6.57 (measured)
- Relative density (20°C): 0.931-0.939 g/cm3
- Storage condition of test material: Room temperature, dry, well ventilated. Keep container closed and filled, keep away from heat sources, protect against high temperature. Do not re-use empty container.
- Storage stability: stable under normal conditions, avoid oxidizing materials
Analytical monitoring:
yes
Details on sampling:
- Concentrations: At test start and at each renewal samples of all treatments were taken from the test solution preparation just before distributing it to the 10 replicates. At the renewals samples of the aged test solution were taken from each vessel, pooled per concentration and measured.
- Sampling method: At the start and at each renewal sub-sample from each freshly prepared test solution were taken for chemical analysis. Additionally pooled samples were taken from each aged test series/concentration. The samples for analysis were filled into 40 mL sample vials. The vials were filled up to the brim to prevent the loss of Muscenone dextro into the headspace of the vials.
For sample preparation, aliquot samples ranging from 0.1 to 5.0 mL were pipetted into 8 mL test tubes, sample volumes less than 5 mL were filled up to 5 mL with purified water. Then 100 µL of the IS working solution and 400 µL cyclohexane were added. The mixtures were shaken thoroughly by hand and additionally for 10 min on a horizontal shaker (about 200 strokes/min). For a rapid phase separation the tubes are centrifuged at 2000 rpm for 2 minutes. The organic supernatants (250 µL aliquots) were transferred into the autosampler vials using disposal Paster pipettes, aliquots of 1µL were analysed by GC/MS.
- Sample storage conditions before analysis: no data
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg (214 µL) of test substance were given into 2 L dilution water in a 2 L flask with a drain port near the bottom. The preparation was stirred at room temperature for 48 h avoiding very strong stirring due to avoiding the generation of fine droplets. After 48 hours the preparation was left for around 18 hours (overnight) to enable droplets to move to the surface of the solution. Then, 200 mL of the aqueous phase were drained off and discarded. The following 600 mL were used for the test preparation without filtration as the highest test concentration. Because adsorption of the test item on filter material cannot be excluded, it is preferred to use test solutions without filtration. The next 1000 mL were used to prepare the dilution series for the further test concentrations. The original eluate was diluted with dilution water by a factor of 2 to obtain the other 4 treatments.
- Eluate: dilution water
- Differential loading: no data
- Controls: yes, the control consists of dilution water only.
- 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 data

The test item is a liquid of poor water solubility. Since the use of a dispersant should be avoided, test solutions were prepared in dilution water as described above to obtain the maximum dissolved concentration of the test substance.

The mean solubility of 1120.8 μg test item/L was determined under the test conditions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain/clone: no data
- Justification for species other than prescribed by test guideline: not applicable
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of hte Fraunhofer IME.
- Age of parental stock (mean and range, SD): 4-24 hours old at test start.
- Feeding during test: yes.
- Food type: suspensions of unicellular alga Desmodesmus subspicatus
- Amount: about 7 to 15 mg C/L
- Frequency: daily

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY).
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no data

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: no data
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
none
Post exposure observation period:
No
Hardness:
total hardness = 1.0 mmol/L
Test temperature:
With 20.4-21.0°C throughout the test the permitted range of 18-22°C (with a variance of less than 2°C) was maintained.
pH:
The pH values throughout the test were within a range of 8.0 - 8.7 at all treatment levels.
Dissolved oxygen:
The oxygen saturation was between 6.4 mg/L and 9.5 mg/L.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg test item/L.
Measured concentrations: 21.0, 40.6, 89.8, 155.0 and 271.7 µg test item/L (corresponding with 30, 29, 32, 27.6 and 24.2% of the nominal concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass beakers
- Type (delete if not applicable): the containers were covered with glass panes
- Material, size, headspace, fill volume: glass, 50 mL
- Aeration: without aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): test solution was exchanged daily.
- 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: purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: 2.0-2.2 mmol/L
- Ca/mg ratio: no data
- Conductivity: 261.2-273.7 µS/cm
- Salinity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: the light intensity did not exceed 15-20 µE/(m²*s) or 1125-1500 lx.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Effects on growth (adult length at test termination) and reproductive performance were investigated.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: Not applicable
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
232 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
(parental survival)
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
165 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(length on day 21)
Remarks on result:
other: 95% CL: 145-181 µg a.i./L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
192 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: cumulative offspring per female
Remarks on result:
other: 95% CL: 185-197 µg a.i/L
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
121 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: intrinsic rate of population increase
Remarks on result:
other: 95% CL: 40-160 µg a.i./L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
155 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
(parental survival)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
89.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(length on day 21)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
89.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: age at first brood
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
155 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: cumulative offspring per female
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
89.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: intrinsic rate of population increase
Details on results:
- Test item concentration:
The mean measured test item concentrations of the freshly prepared test solutions of the active ingredient (initial concentrations) were between 100% and 128.4% of nominal (based on the solubility under test conditions). During the time interval until renewal of the test solution, active ingredient concentrations decreased considerably to 2.7-3.6% of nominal. The average time weighted means (TWM) of measured initial and aged concentrations at test solution renewal were 21.0, 40.6, 89.8, 155.0 and 271.7 µg test item/L, corresponding with 30.0, 29.0, 32.0, 27.6 and 24.2% of the nominal concentrations.

- Survival, growth and reproduction data:
See table 6.1.4/1 in "Any other information on results incl. tables"
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
For each endpoint, the NOEC, LOEC and if possible the EC50 and EC10 were determined. Calculations were performed with the computer software ToxRat. A NOEC was calculated by using ANOVA following by Williams' test or an appropriate non-parametric test suggested by the ToxRat program. When the test results showed a loading-response relationship, the data were analysed by regression to determine the EC50 including the 95% confidence interval as well as the EC10 using Probit-analysis assuming log-normal distribution of the values.

Table 6.1.4/1: Survival, growth and reproduction data

Concentration

Parental survival

Growth (length on Day 21)

Age of first brood

Cumulative offspring per female

Intrinsic rate of population increase

TWM

(µg test item/L)

(%)

Mean +/- SD (days)

Mean +/- SD (days)

Mean +/- SD (Ind.)

Mean +/- SD (Ind./day)

Control

100

4.85 +/- 0.32

8.5 +/- 0.9

85.4 +/- 8.2

0.358 +/- 0.041

21.0

100

4.86 +/- 0.27

8.7 +/- 1.1

81.0 +/- 14.1

0.337 +/- 0.035

40.6

100

4.88 +/- 0.30

8.5 +/- 1.1

96.2 +/- 19.4

0.350 +/- 0.040

89.8

100

4.71 +/- 0.33

8.9 +/- 1.0

85.2 +/- 9.4

0.326 +/- 0.025

155.0

100

4.48 +/- 0.17*

10.1 +/- 0.7*

84.4 +/- 8.0

0.299 +/- 0.016*

271.7

50

3.41 +/- 0.44*

13.3 +/- 1.6*

32.0 +/- 20.6*

0.162 +/- 0.057*

Table 6.4.4/2: Effect summary table, based on concentrations (µg a.i./L) calculated from time weighted mean concentrations

Concentration

Parental survival

Growth (length on day 21)

Age at first brood

Cumulative offspring per female

Instrinsic rate of increase

EC50

(95% CL)

272

(n.d.)

> 272

(> 272)

/

253

(252 - 255)

258

(213 - >272)

EC10

(95% CL)

232

(n.d.)

165

(145 - 181)

/

192

(185 - 197)

121

(40 – 160)

NOEC

155.0*

89.8

89.8

155.0

89.8

n.d. = not determined due to mathematical reasons; * Value assessed by expert knowledge

Validity criteria fulfilled:
yes
Conclusions:
The test substance has a chronic adverse effect on Daphnia magna. The lowest EC10 value was determined at 121 µg test item/L TWM, for the intrinsic rate of population increase.
Executive summary:

A 21 -day semi-static exposure to the test substance at different concentrations with renewal of the test solutions three times a week was conducted according to OECD guideline 211. Untreated control replicates were run in parallel. Each treatment group consisted of 10 replicates with one daphnid each (individual exposure). Effects on growth (adult length at test termination) and reproductive performance were investigated. Test item concentrations were measured at fresh and aged test solutions. The mean solubility of 1120.8 μg test item/L was determined under the test conditions.

The average time weighted means (TWM) of measured initial and aged concentrations at test solution renewal were 21.0, 40.6, 89.8, 155.0 and 271.7 µg test item/L, corresponding with 30.0, 29.0, 32.0, 27.6 and 24.2% of the nominal concentrations.

TWM test concentrations up to 271.7 µg test item/L did not affect survival of adults statistically significant. However, with 50% mortality at test end in the highest concentration a relevant effect occurs (NOEC survival = 155 µg test item/L TWM). The EC10 and EC50 survival of 232 and 272 µg test item/L TWM were determined, respectively. No clinical sign was observed for the survived individuals.

Adult growth (body length) was affected significantly starting at 155 µg test item/L TWM. The NOEC adult growth was found to be 89.8 µg test item/L TWM. The EC10 and EC50 adult growth of 165 and > 272 µg test item/L TWM were determined, respectively.

The age at first brood was affected significantly starting at 155 µg test item/L TWM. The NOEC first brood was found to be 89.8 µg test item/L TWM.

Adult reproduction was affected significantly starting at 271.7 µg test item/L TWM. The NOEC reproduction was found to be 155 µg test item/L TWM. The EC10 and EC50 reproduction of 192 and 253 µg test item/L TWM were determined, respectively.

Intrinsic rate of population increase was affected significantly starting at 155 µg test item/L TWM. The corresponding NOEC was found to be 89.8 µg test item/L TWM. The EC10 and EC50 intrinsic rate of population increase of 121 and 258 µg test item/L TWM were determined, respectively.

In conclusion, the test substance has a chronic adverse effect on Daphnia magna. Adult growth, age of first brood, and intrinsic rate of population increase were affected starting at 155 µg test item/L time weighted means (TWM) (LOEC). The relevant NOEC adult growth, age of first brood and intrinsic rate of population increase is 89.8 µg test item/L TWM. The relevant EC50 (reproduction) is 253 µg test item/L TWM. Finally, the lowest EC10 value was determined at 121 µg test item/L TWM, for the intrinsic rate of population increase.

Description of key information

OECD Guideline 211, GLP, key study, validity 1:

Lowest 21d-EC10 (intrinsic rate of population increase) = 121 µg/L (time weighted mean measured concentrations)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
121 µg/L

Additional information

One key study is available to assess the long-term toxicity of the substance to aquatic invertebrates.

This study (Fraunhofer, 2009) was performed according to OECD Guideline 211 with GLP certificate. A 21 -day semi-static exposure to the test substance at different concentrations with renewal of the test solutions three times a week was conducted. Untreated control replicates were run in parallel. Each treatment group consisted of 10 replicates with one daphnid per replicate. Effects on growth (adult length at test termination) and reproductive performance were investigated. Test item concentrations were measured at fresh and aged test solutions. The mean solubility of 1120.8 μg test item/L was determined under the test conditions. The average time weighted means (TWM) of measured initial and aged concentrations at test solution renewal were 21.0, 40.6, 89.8, 155.0 and 271.7 µg test item/L, corresponding to 30.0, 29.0, 32.0, 27.6 and 24.2 % of the nominal concentrations. The lowest chronic EC10 value, based on intrinsic rate of population increase, was determined at 121 µg test item/L, based on time weighted mean measured concentrations.