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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 march 2010 to 15 july 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to standardised guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The concentrations of the test item GUETOL were analyzed in the duplicate test media samples from the nominal concentrations of 3.2 to 100 mg/L from both sampling times (0 and 48 hours). The samples of the nominal test concentration of 1.0 mg/L were not analyzed since the concentration was below the 48 hour NOEC determined in this test.

- Sampling method:
For the determination of the actual test item concentrations, duplicate samples were taken from each treatment before the test start and at the end of the test after 48 hours.
For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
From the control, only one of the duplicate samples was analyzed per sampling time.

- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (non-GLP), the test item was found 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: The test medium of the highest nominal concentration of 100 mg/L was prepared in a completely filled and tightly closed stirring vessel by dissolving 58.6 mg of the test item in 580 mL of test water stirring intensely for 10 minutes at room temperature. Adequate volumes of this test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before introduction of the daphnids (i.e., start of the test).
- Eluate: No
- Differential loading: No
- Controls: a control (test water without test item) was tested in parallel; four replicates.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The appearance of the test media was visually recorded at the start of the test and after 24 and 48 hours. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, Clone 5
- Source: originally supplied by the University of Sheffield/UK in 1992.
- Age at study initiation): At the start of the test, the test organisms were 6-24 hours old and were not be first brood progeny.
- Method of breeding: The clone (defined by the supplier as clone 5) is successfully bred in Harlan Laboratories under temperature and light conditions identical to those of the tests. The cultivation of the parental daphnids is performed in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness). During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
- Feeding during test: no

ACCLIMATION: not applicable

Study design

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

Test conditions

Hardness:
250 mg/L as CaCO3
Test temperature:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20-21 °C.
pH:
At the beginning and end of the test period, the pH value of the test media was in the range of 7.8 to 7.9
Dissolved oxygen:
The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated. At the beginning and end of the test period, the dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L,
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32, and 100 mg/L.
Measured: At the start and at the end of the test, the analytically determined concentrations of the test item in the test media were between 96 and 103% of the nominal values.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50-mL glass test tubes
- Type (delete if not applicable): closed; The test tubes were made tight with glass stoppers to avoid losses of the volatile substance.
- Material, size, headspace, fill volume: glass/ 50 mL/ 0/about 50 mL of test medium
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): NA
- Biomass loading rate: 1 daphnia per mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations
CaCl2 × 2H2O: 294 mg/L
MgSO4 × 7H2O: 123 mg/L
NaHCO3: 65 mg/L
KCl: 5.8 mg/L

- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and at the end of the test, the pH values, dissolved oxygen concentrations and water temperature were determined at each treatment.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period.
- Light intensity: approximately between 520 and 680 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 14-20 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 14-20 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: not reported
- Observations on body length and weight: NA
- Other biological observations: not reported
- Mortality of control: no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.
- Effect concentrations exceeding solubility of substance in test medium: NA
Results with reference substance (positive control):
The result of the latest positive control test in March 2010 (48-hour EC50: 0.43 mg/L,study C86933) proved the sensitivity of the test organisms and the validity of the test design (internal historical range of 48-hour EC50 from 2000 to 2010: 0.43‑1.1 mg/L).
Reported statistics and error estimates:
The 24- and 48-hour EC50 and the 95% confidence limits were calculated by Probit Analysis using linear maximum likelyhood regression.

Any other information on results incl. tables

At the start and end of the test, the analytically determined concentrations of the test item in the test media were between 96 and 103% of the nominal values (see analytical results and Table 2 in the corresponding appendix). Thus, the correct dosing of the test item GUETOL was confirmed. Furthermore, it has been demonstrated that the test item was stable during the test period of 48 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

Effect of GUETOL on the Mobility of Daphnia magna in the definitive test

Nominal
test item
concentration

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

 

No.

%

No.

%

Control

20

0

0

0

0

1.0

20

0

0

0

0

3.2

20

0

0

0

0

10

20

1

5

1

5

32

20

20

100

20

100

100

20

20

100

20

100

 

Effect of GUETOL on the Mobility of Daphnia magna in the range finding test 

Nominal

test item concentration

 

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

 

No.

%

No.

%

Control

10

0

0

0

0

1.0

10

0

0

1

0

10

10

10

100

10

10

100

10

10

100

10

100

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids showed immobilization or other signs of disease or stress. Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.
Conclusions:
This study is classified as Klimisch 1 and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.
Based on the results of this study, guetol would be considered as harmful for aquatic invertebrates.
Executive summary:

The acute toxicity of the test item GUETOL to Daphnia magna was determined in a 48‑hour static test (Harlan, 2010), according to the EU C.2, and the OECD 202.

The nominal test item concentrations tested were 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control group (test water without test item) was tested in parallel. The test was performed using glass tubes completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item (closed system).

At the start and at the end of the test, the analytically determined concentrations of the test item in the test media were between 96 and 103% of the nominal values. Thus, the correct dosing of the test item GUETOL was confirmed. Furthermore, it has been demonstrated that the test item was stable during the test period of 48 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

 

The biological test result was as follows:

– 48-hour EC50:

16 mg/L (95%CL: 14 and 20 mg/L)

Based on the results of this study, guetol would be considered as harmful for aquatic invertebrates.