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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:

Chemical name: Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane
CAS No.: 17557-23-2
EC / List no.: 701-333-0
Analytical monitoring:
yes
Details on sampling:
Based on the results obtained during analytical method validation (Study number:
555-100-6268) the test item was expected to be stable for the duration of 48 hours in ISO Medium. For determination of the test item concentrations, samples were taken from the test item treatment and control groups at the start (prior to distributing of the test solutions to the test vessels) and at the end of the experiment. The samples taken (1 mL/replicate) were sent to individual analysis.
Vehicle:
no
Details on test solutions:
The test item was completely and evenly diluted in ISO medium by mechanical dispersion without the use of any solubilising agent in the testing laboratory.
0.0257 g of test item was diluted in 514 mL of the dilution water (ISO medium; see section 5.4) in order to give the nominal loading rate of 50 mg/L of the test item. This test item stock solution was handled in ultrasonic bath to have a clear solution (for about 20 min). The further test item concentrations were prepared from this test item stock solution (TISS) as shown in the following table.

Table 2: Test Solution Preparation
Test item concentration
(mg/L) Volume
(mL) Dilutions
50 514 TISS
25 250 125 mL TISS ad 250 mL (ISO medium)
12.5 250 62.5 mL TISS ad 250 mL (ISO medium)
6.25 250 31.25 mL TISS ad 250 mL (ISO medium)
3.125 250 15.625 mL TISS ad 250 mL (ISO medium)
After the formulation procedure the test animals were immediately introduced into the test solutions. 
Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection)
2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in every group (test concentrations and untreated control), divided into 4 replicates (5 animals per replicates).
Age of animals: Less than 24 h old at the start of exposure period.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The measured hardness of the ISO Medium used in the test was determined to be 249.2 mg/L (as CaCO3).
Test temperature:
The test temperature was in the range of 21.4 – 21.8 °C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 21.0 – 21.9 °C.
pH:
The pH of the test solutions was not adjusted and not varied by more than 1.5 units in any one test. The measured pH was in the range of 6.99 – 7.60 during the test.
Dissolved oxygen:
The measured dissolved oxygen concentration was in the range of 7.64 – 8.41 mg/L during the test.
Nominal and measured concentrations:
measured
Details on test conditions:

The test vessels were kept in a climate chamber under controlled environmental conditions during the test and test solutions were not aerated. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the experiment. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.
Test type: Static test. Based on the results obtained during analytical method validation (Study number: 555-100-6268) the test item was expected to be stable for the duration of 48 hours in ISO Medium.

6.2       Test Item Concentrations in the Definitive Test


Based on the results of the preliminary range-finding test five test concentrations in a geometric series with a spacing factor of 2.0 and one control were included in the main test.

The following nominal concentrations were tested: 3.125, 6.25, 12.5, 25 and 50 mg test item / L.

Mean of the measured test item concentrations did not remain within ± 20 % of the nominal by the end of the experiment, therefore the biological results are based on the geometric mean of the measured concentrations. The corresponding calculated test item concentrations were 2.57, 5.03, 10.21, 19.87 and 39.27 mg test item/L.



6.3       Performance of the Test


Twenty animals, divided into four replicates (glass beaker; volume app. 50 mL) of five animals each (at least 4 mL test solution/animal) were exposed to the test concentrations or included as control over a period of 48 hours in a static test. The test animals were not fed during the test. Each test vessel contained approximately 40 mL of test solution or control and was uniquely identified with study number, test group and replicate. The test solutions were not aerated during the exposure period.

Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition to immobility, any abnormal behaviour or appearance is reported.

The environmental conditions were measured as described above (Section: 5.5).

Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 6.21 mg/L
95% CI:
>= 4.49 - <= 7.64
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC10
Effect conc.:
ca. 4.96 mg/L
95% CI:
>= 3.28 - <= 6.31
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 9.51 mg/L
95% CI:
>= 7.74 - <= 11.66
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 2.57 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Validity of the Study

Immobilisation was 0.0 % (< 10 %) in the control group and none of the daphnids showed signs of disease or stress. The dissolved oxygen concentration measured during the test in control and test vessels was in the range of 7.64 – 8.41 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study is considered as valid.

10.2 Analytical Results

The concentrations of the test item were analysed at the start and at the end of the experiment.
Signal intensities measured for the untreated control samples were below the quantification limit (i.e. no interfering component was detected in the untreated control samples).
Mean measured concentrations of the test item were in the range of 86.8 – 91.2 % of the nominal concentrations at the start of the exposure period and between 70.4 – 74.2 % of the nominal concentrations at the end of the exposure period. Therefore, the biological results are based on the geometric mean of the measured concentrations.
Detailed description of the analytical part of the study (method, determination, results) is attached to the Report as Appendix III.

10.3 Biological Results

No immobilisation was observed either in the control group or at the lowest test item concentration of 2.57 (nominal 3.125) mg/L. In addition to immobility, no abnormal behaviour or appearance of test animals was detected in these test groups. Statistical significantly difference was calculated in the test item concentration range of 5.03 – 39.27 (nominal 6.25 – 50) mg test item/L (Step-down Cochran-Armitage Test; α = 0.05).
. Accordingly, the 48-h NOEC was determined to be 2.57 (nominal 3.125) mg/L, while the 48-h LOEC was determined to be 5.03 (nominal 6.25) mg test item/L.
The 48-h-EC50 was calculated to be 9.51 mg/L (see Appendix II).

All biological results are based on the geometric mean of the measured concentrations.

Table 5: Immobilisation of the Test Animals (Summary Table)
Nominal concentration
(mg/L) Mean measured concentration
(mg/L) No. of
Daphnia
tested No. of immobilised
Daphnia after % of immobilised
Daphnia after
24 h 48 h 24 h 48 h
Control - 20 0 0 0 0
3.125 2.57 20 0 0 0 0
6.25 5.03 20 0 2 0 10
12.5 10.21 20 12 12 60 60
25 19.87 20 14 18 70 90
50 39.27 20 19 20 95 100

Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Reported statistics and error estimates:
The 48-h ECx values and their 95 %-confidence limits were calculated by Probit analysis (statistical software program ToxRatPro Version 3.3.0). NOEC and LOEC values were determined by Step-down Cochran-Armitage Test (α = 0.05; statistical software program ToxRatPro Version 3.3.0).
Validity criteria fulfilled:
yes
Conclusions:
All validity criteria were met and therefore the study is considered as valid.

Biological endpoints of the study are summarised below:

Summary of the Biological Endpoints (same a Table 1)
Endpoint Concentration [mg/L]
based on measured concentrations
48 h-EC10 4.96
95 % confidence limits 3.28 – 6.31
48 h-EC20 6.21
95 % confidence limits 4.49 – 7.64
48 h-EC50 9.51
95 % confidence limits 7.74 – 11.66
48 h-NOEC 2.57 (nominal: 3.125)
48 h-LOEC 5.03 (nominal: 6.25)

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane had significant toxic effects on the mobility of Daphnia.
The 48-h EC50 value was determined to be 9.51 mg/L.
The 48-h NOEC was determined to be 2.57 (nominal: 3.125) mg/L.
The 48-h LOEC was determined to be 5.03 (nominal: 6.25) mg/L.
All validity criteria were met. The results are based on the geometric mean of the measured test item concentrations.

Executive summary:

Acute toxicity of the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane was assessed on Daphnia magna in a 48-hour acute immobilisation test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a static test.


The test animals were exposed to aqueous test media containing the test item for 48 hours at a range of nominal concentrations as 3.125, 6.25, 12.5, 25 and 50 mg/L plus a control. Concentration range was chosen based on results obtained in a non-GLP pre-test.


The quantification of the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentrations and from the control at the start and at the end of the experiment and analysed by LC-MS/MS method.


Mean of the measured test item concentrations did not remain within ± 20 % of the nominal at the end of the test, therefore the biological results are based on the geometric mean of the measured concentrations. The corresponding calculated test item concentrations were 2.57, 5.03, 10.21, 19.87 and 39.27 mg test item/L.


 


Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the test concentrations and to the control medium. Each test vessel contained approximately 40 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.


 


The immobilisation of the test animals was observed 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of the experiment. All measured values remained within the acceptable ranges.


 


After 48 hours exposure no immobility was observed in the control group and at the lowest test concentration of 2.57 (nominal 3.125) mg/L. Statistically significant immobilization compared to the control was observed in the concentration range of 5.03 – 39.27 (nominal 6.25 – 50) mg test item/L. Accordingly, the 48h NOEC was determined to be 2.57 (nominal 3.125) mg/L, while the 48-h LOEC was determined to be 5.03 (nominal 6.25) mg test item/L.


 


The 48-h-EC50 was calculated to be 9.51 (95 % confidence limits: 7.74 – 11.66) mg/L.


 


The 48-h ECx values were calculated by Probit analysis (statistical software program ToxRatPro Version 3.3.0). NOEC and LOEC values were determined by Step-down Cochran-Armitage Test (α = 0.05; statistical software program ToxRatPro Version 3.3.0).


All validity criteria were met and therefore the study is considered as valid (see section 10.1)


 


The biological endpoints are summarized below in Table 1.


 


 


Table 1: Summary of the Biological Endpoints










































Endpoint



Concentration [mg/L]
based on measured concentrations



48 h-EC10



4.96



95 % confidence limits



3.28 – 6.31



48 h-EC20



6.21



95 % confidence limits



4.49 – 7.64



48 h-EC50



9.51



95 % confidence limits



7.74 – 11.66



48 h-NOEC



2.57 (nominal: 3.125)



48 h-LOEC



5.03 (nominal: 6.25)



 


In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane had significant toxic effects on the mobility of Daphnia.


The 48-h EC50 value was determined to be 9.51 mg/L.


The 48-h NOEC was determined to be 2.57 (nominal: 3.125) mg/L.


The 48-h LOEC was determined to be 5.03 (nominal: 6.25) mg/L.


All validity criteria were met. The results are based on the geometric mean of the measured test item concentrations.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
ca. 9.51 mg/L

Additional information