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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
Study period:
2018-05-17 to 2018-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline; EU Method; GLP
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: TEMPIC
Synonym: THIOCURE TEMPIC
Chemical Name: Tris[2-(3-mercaptopropionyloxy)ethyl]isocyanurat
CAS No.: 36196-44-8
Batch No.: 0061488
Purity: 95.9 % according to certificate of analysis
Manufacturing Date: December 21, 2017
Certificate of Analysis Date: December 20, 2017
Aggregate State at Room Temperature: Liquid
Colour: Light yellow
Expiry Date: December 20, 2018
Storage Conditions at Test Facility: At 4 +/- 4 °C, in the dark
Analytical monitoring:
yes
Remarks:
LC-MS/MS
Details on sampling:
Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the aged test media of the only test concentration and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling without any sample treatment.
Vehicle:
no
Details on test solutions:
Test Concentration: 10 mg test item/L and a control, corresponding to following geometric mean measured concentrations of the test item: 2.78 mg test item/L and a control. Control: In the control, test water was used without addition of test item.
Dosage of Test Item: The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 10 mg test item/L was prepared by suspending 20 mg and 20 mg test item in 2000 mL and 2000 mL test water. The stock suspension was stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter).
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 5 to 23.5 hours old Sex: Female
Origin: The daphnids introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium (see 6.5). The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate
Test temperature:
21 °C at test start;
21 °C at test end
pH:
8.1 to 8.3 in the freshly prepared media; 7.9 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
4.4 to 9.0 mg/L in the freshly prepared media; 8.5 to 8.8 mg/L in the aged test media
Nominal and measured concentrations:
10 mg test item/L and a control; corresponding to following geometric mean measured concentrations of the test item: 2.78 mg test item/L and a control.
Details on test conditions:
Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature: The water temperature, pH-values and the dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group.
Water Temperature: 20.8 °C in the freshly prepared media; 20.6 to 20.7°C in the aged test media
pH-Values: 8.1 to 8.3 in the freshly prepared media; 7.9 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 4.4 to 9.0 mg/L in the freshly prepared media; 8.5 to 8.8 mg/L in the aged test media Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 350 to 960 lux (measured once during the test).
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 2.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 2.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of 2.78 mg test item/L.
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate performed in June 2017 (study code 88298220), the EC50 after 24 hours was determined to be 1.58 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
No statistical analysis was performed. The EC50 could not be statistically quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.
Validity criteria fulfilled:
yes
Remarks:
, Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.7 mg O2/L in the control and test vessels at the end of the test.
Conclusions:
The toxic effect of the test item TEMPIC to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be ≥ 2.78 mg test item/L. The 48-hour LOEC was determined to be > 2.78 mg test item/L and the 48-hour EC50 value was determined to be > 2.78 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part.
All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or initial concentrations during the test.
Executive summary:

Title:

TEMPIC: Acute Toxicity to Daphnia magna in a Semi-Static 48-hour Immobilisation Limit Test

Purpose:

 The purpose of this study was to determine the influence of the test item TEMPIC on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test for 48 hours to the only concentration of nominal 10 mg/L under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines:

-Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated May 30, 2008

-OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

-OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

-SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

Test Item: TEMPIC; batch no.: 0061488; purity: 95.9 %, according to certificate of analysis

Test Species: Female Daphnia magna, clone 5; 5 to 23.5 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design: This study encompassed two treatment groups (one test item concentration at nominal 10 mg/L and one control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentration: 10 mg test item/L and a control corresponding to following geometric mean measured concentrations of the test item: 2.78 mg test item/L and a control.

Test Conditions: Water temperature: 20.6 to 20.8 °C; pH value: 7.9 to 8.3; dissolved oxygen concentration: 4.4 to 9.0 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 350 to 960 lux; and thus were within the ranges requested by guideline OECD 202

Results

Biological test results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of 2.78 mg test item/L.

Analytical test results:

The quantification of the test item TEMPIC in the test samples was performed using liquid chromatography with MS/MS detection. In the freshly prepared test media at the start of the test and at the renewal of the test media 35 % of the nominal test concentrations were found (average of all replicates of the only concentration). In the aged test media after 24, and 48 hours test duration, 22 % of the nominal value was determined (average of all replicates of the only concentration).

Conclusion:

The toxic effect of the test item TEMPIC to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be ≥ 2.78 mg test item/L. The 48-hour LOEC was determined to be > 2.78 mg test item/L and the 48-hour EC50 value was determined to be > 2.78 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or initial concentrations during the test.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Description of key information

OECD TG 202 study conducted with TEMPIC.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.78 mg/L

Additional information

A NOEC 2.78 mg/L could be reliable reported from this study.