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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 22-Jun-2010 to 15-Oct-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item 4-TBC were analyzed in duplicate test media samples from the nominal concentrations of 0.22, 0.46, 1, 2.2, 4.6 and 10 mg/L.
- Sampling method: Duplicates samples were taken from each treatment at the start and end of each test medium renewal period.
- Sample storage conditions before analysis: All the samples were analyzed directly after sampling without storage.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared daily at the start of the test and prior each test medium renewal by completely dissolving 100 mg of test item (dosed amounts: 100.0 and 100.2 mg) in 1000 mL of test water under stirring for 15 minutes at room temperature. This stock solution was used in a series of dilutions to prepare the test media of all test item concentrations. The test media were prepared just before introduction of the daphnids (i.e., start of the test and prior to the test medium renewal)
- Eluate: no
- Differential loading: no
- Controls: a blank (test water without test item) was tested in parrallel; four replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The appareance of the test media was visually recorded at the start and end of each test medium renewal period. 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: Water flea
- Strain: clone defined by the supplier as clone 5
- Source: 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
- Weight at study initiation (mean and range, SD): no data
- Method of breeding: The clone (defined by the supplier as clone 5) is bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the test. 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, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions 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
- Acclimation period: not applicable


Study design

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

Test conditions

Hardness:
2.5 mmol/L (= 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 21°C.
pH:
At the beginning of each test period, the pH value of the test media was 6.0 and at the end of each test medium renewal period, the pH value of the test media was in the range of 6.8 to 7.5.
Dissolved oxygen:
At the beginning and end of the test medium renewals periods, the dissolved oxygen concentrations in the test media and control were at least 7.9 mg/L.
Salinity:
not applicable
Nominal and measured concentrations:
nominal concentrations: 0, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
measured concentrations: 0, 0.18, 0.41, 0.94, 1.97, 4.47 and 9.94 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type: closed; The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solution.
- Material, size, headspace, fill volume: vessels filled with 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.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (semi-static test)
- Renewal rate of test solution (frequency/flow rate): medium renewal after 24 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium of the corresponding concentration.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 2mL of medium per daphnia

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

- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Na/K ratio: 10:1 (based on molarity)
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and end of each test medium renewal period, the pH values, dissolved oxygen concentrations and water temperature were determined at each treatment. The appearance of the test media was visually recorded at the same time.
OTHER TEST CONDITIONS
- Adjustment of pH: yes. The pH of the test media was adjusted to 6.0 with a 1 M hydrochloric acid solution.
- Photoperiod: a 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:
The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized. Additionally, the test animals were observed for toxic effects including discoloration of the test organisms and abnormal behavior.
The NOEC, EC0 and EC100 were determined directly from the raw data.
The 24- and 48-hour EC50 and the 95% confidence limits were calculated by Probit Analysis.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study:
- Test concentrations: 0, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes, the test concentrations were based on results of a range-finding test of a pre-experiment to determine water solubility of the test item (non-GLP); 100% mortality for the concentration 10 mg/L and 20% mortality for the concentration 1.0 mg/L, pH 7.8.
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 0.63 - 1.3 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 0.10 - 1.1 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
< 0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
During the first 24 hours of the test, no immobilization test organisms were determined in the control. At the mean measured test concentrations of 0.18 to 4.5 mg/L, the immobilization was between 20 and 90%. At the highest test concentration of 9.9 mg/L, all test organisms were found to be immobile after 24 hours.

After 48 hours of exposure, no immobilized test organisms were determined in the control. At the concentrations of 0.18 to 0.94 mg/L, the immobilization was between 30 and 50%. From the concentration of 1.97 mg/L onwards, all daphnids were found to be immobile. Except immobility, no other adverse effects were observed in the test concentrations at observation after 48 hours. (see Table 1)

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

Results with reference substance (positive control):
The result of the lastest positive control test of 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:
In the analyzed test medium samples from the start of the test medium renewal periods, the measured test item concentrations ranged between 98 and 106% of the nominal values. This shows the correct dosage of the test item. During the test medium renewal periods of 24 hours, a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 61 to 94% of the nominal values were found.

The biological results were based on mean measured concentrations calculated as geometric means of all measurements (see Table 2)

Any other information on results incl. tables

Table 1: Effect of 4 -tert-butylpyrocatechol on the mobility of Daphnia magna in the definite test for each concentration, pH 6.0

Nominal
test item
concentration

Mean measured concentration

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

(mg/L)

 

No.

%

No.

%

Control

--

20

0

0

0

0

0.22

0.18

20

4

20

6

30

0.46

0.41

20

  4 +

20

8

40

1.0

0.94

20

  8 +

40

10

50

2.2

1.97

20

14

70

20

100

4.6

4.47

20

  18 +

90

20

100

           10

9.94

20

20

100

20

100

+ One test organism at this concentration was trapped at the water surface.

Table 2: Mean measured test item concentration , pH 6.0

Nominal test item
concentration (mg/L)

Mean measured test item concentration

(mg/L)

% of nominal

0.22

0.18

79.7

0.46

0.41

88.3

1.0

0.94

93.8

2.2

1.97

89.7

4.6

4.47

97.1

10

9.94

99.4

Table 3: Effect of 4 -tert-butylpyrocatechol on the mobility of Daphnia magna in the range finding test, pH 7.8

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

0.10

20

0

0

0

0

1.0

20

1

10

2

20

10

20

10

100

10

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
control immobilization rate < 10% and dissolved oxygen concentration at the end of the test > 3 mg/L in the control and test vessels
Conclusions:
Based on the results of this study, 4-tert-butylpyrocatechol would be considered as very toxic for aquatic invertebrates.
Executive summary:

In a 48 -hour acute toxicity study (Harlan, 2010), Daphnia magna were exposed to 4 -tert- butylpyrocatechol (4 -TBC) under semi-static conditions according to the Commission Regulation (EC) n° 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, n°202 (2004).

Daphnids were exposed to control and test item at nominal concentration of 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L. The pH of the test medium was set at pH 6.0 at start of the experiment in order to increase the stability of test item (see Hydrolysis, section 5.1.2) and better assess the intrinsic toxicity of 4 -TBC (see Table 3).

In the analyzed test medium samples from the start of the test medium renewal periods, the measured test item concentrations ranged between 98 and 106% of the nominal values. During the test medium renewal periods of 24 hours, a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 61 to 94% of the nominal values were found.

The biological results were based on mean measured concentrations calculated as geometric means of all measurements:

Nominal test item
concentration (mg/L)

Mean measured test item concentration

(mg/L)

% of nominal

0.22

0.18

79.7

0.46

0.41

88.3

1.0

0.94

93.8

2.2

1.97

89.7

4.6

4.47

97.1

10

9.94

99.4

Mortality / immobilization were observed daily.

After 48 hours of exposure, no immobilized test organisms were determined in the control. At the concentrations of 0.18 to 0.94 mg/L, the immobilization was between 30 and 50%. From the concentration of 1.97 mg/L onwards, all daphnids were found to be immobile. Except immobility, no other adverse effects were observed in the test concentrations at observation after 48 hours.

The 48 -hour EC50 was 0.48 mg/L with 95% confidence limits of 0.10 and 1.1 mg/L.

The 48 -hour NOEC was < 0.18 mg/L, since a statistically significant effect was observed at this test concentration.

In summary, the biological test results were as follows:

- 24-hour EC50:

0.94 mg/L (95% CL.: 0.63 – 1.3 mg/L)

- 48-hour EC50:

0.48 mg/L (95% CL.: 0.10 – 1.1 mg/L)

- 48-hour NOEC:

< 0.18 mg/L

- 48-hour EC100:

1.97 mg/L

Based on the results, 4 -TBC was very toxic for aquatic invertebrates.

This study is classified as acceptable and satisfies the guideline requirements for acute daphnids toxicity study.