diethyl pyridine-2,4-dicarboxylate

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-06-13 to 2016-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 236 " Fish Embryo Acute Toxicity (FET) Test ", adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

MEXORYL SBU

IUPAC Name:

MEXORYL SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Test material form:

other: solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: 0156553- Expiration date of the lot/batch: 2017-05-06- Purity test date: 2015-06-16STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature (20 ± 5 °C), in the darkTREATMENT OF TEST MATERIAL PRIOR TO TESTING: no

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations, sampling method and sample storage conditions before analysis: One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media of all treatment groups were taken on Day 0, Day 1, Day 2 and Day 3 of exposure. For the determination of the stability of the test item, respectively the maintenance of the test item concentrations under the test conditions, duplicate samples of all treatment groups were taken from all aged test medium concentrations (on Day 1, Day 2 and Day 3) and at the end of the last renewal period (on Day 4). For sampling, the test solution of all petri dishes from each concentration was merged. All samples were diluted by a factor of 5 with acetonitrile, apart from the aged test media samples taken on Day 2 where the samples were diluted by factor 2. Additional samples of the control blank and the dilution solvent were taken at each sampling time without any sample treatment.The samples of the aged test media from Day 2 and Day 4 were analysed standby. All other samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. All remaining parts of the samples were stored in a freezer (≤ - 20 °C), protected from light.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)Before the test start and at each test medium renewal a concentrated stock solution of nominal 100 mg test item/L was prepared by dissolving 20.0 mg test item into 200 mL test water by intense stirring for 10 minutes. Then, adequate volumes of this stock solution were mixed into test water to obtain the desired test concentrations.The test media were prepared just before introduction of the test fish embryos (= start of the test and test medium renewal on day 1, 2 and 3). Eggs were immersed in the petri dishes containing the 5 concentrations of the test item, the positive control, the negative control and the MOPS buffer control and the internal plate control at cell stage 4 to 16.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM- Common name: Zebrafish- Source: In-house breeding. Breeding was performed in breeding groups and the Zebrafish eggs were collected in spawn traps.- Age at study initiation (mean and range, SD): Embryos; The cell stage of the embryos introduced at test start ranged from 4 to 16; Quality of the Eggs (Fertilisation rate): The fertilisation rate of the egg batch, determined from a subsample of 100 eggs, was 100 %. - Length at study initiation (length definition, mean, range and SD): /- Weight at study initiation (mean and range, SD): /- Method of breeding: Breeding was performed in breeding groups and the Zebrafish eggs were collected in spawn traps. The holding and breeding of Zebrafish in the laboratories of ibacon are under similar temperature and light conditions. Holding and breeding was performed in Reconstituted Water (ISO Medium, see 6.5).- Feeding during test: NoACCLIMATION no. QUARANTINE (wild caught) no

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Control: 284.8 mg CaCO3/L. Buffer control: 267.0-302.6 mg CaCO3/L. Positive control: 267-284.8 mg CaCO3/L. test items concentrations: 267-302.6 mg CaCO3/L

Test temperature:

From 26.0 to 26.1°C

pH:

Control: 6.5-7.8. Buffer control: 6.5-6.7. Positive control: 6.6-7.9. test items concentrations: 6.5-6.7

Dissolved oxygen:

Control: 94-104% saturation. Buffer control: 96-102% saturation. Positive control: 94-104% saturation. test items concentrations: 94-103% saturation

Salinity:

/

Conductivity:

Control: 634.0-682.0 µS/cm. Buffer control: 735.0-773.0 µS/cm. Positive control: 655.0-665.0 µS/cm. test items concentrations: 737.0-769.0 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 2.5, 1.7, 1.1, 0.7 and 0.5 mg test item/L. Mean measured concentrations: 2.12, 1.45, 0.912, 0.611, 0.415 mg test item/L (mean value of all measured samples per treatment group).

Details on test conditions:

TEST SYSTEM- Test vessel: petri dishes ; The glass petri dishes were not be preconditioned- Type (delete if not applicable): open / closed- Material, size, headspace, fill volume: glass, approximately 20 mL volume with approximately 10 mL of test medium- Aeration: No- Type of flow-through (e.g. peristaltic or proportional diluter): /- Renewal rate of test solution (frequency/flow rate): daily renewal rate- No. of organisms per vessel: 1 embryo per petri dish- No. of vessels per concentration and control (replicates): -20 eggs for each test concentration-20 eggs as positive control -4 eggs in test water with the addition of buffer as internal plate control (applied for all test concentrations and the positive control)-24 eggs in test water as negative control-24 eggs as buffer control- Biomass loading rate: /TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Reconstituted Water (ISO Medium)Analytical grade salts will be added at the following nominal concentrations in deionised water:CaCl2 * 2H2O2.0 mmol/L(= 294 mg/L)MgSO4 * 7H2O0.5 mmol/L(= 123 mg/L)NaHCO30.75 mmol/L(= 65 mg/L)KCl0.075 mmol/L(= 5.8 mg/L)Water hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3Alkalinity:0.8 mmol/LRatio of Ca : Mg = 4 : 1 (based on molarity); Na : K=10 : 1 (based on molarity)The following buffer was added to the test water: 4-morpholinepropane sulphonic acid (MOPS buffer) 10mMFor the negative control no buffer was added to the test water. In the negative control, test water was used without addition of test item, MOPS buffer, the reference item or the test item. In the buffer control the test water with MOPS buffer addition, without addition of the reference test item of the test item was used.In the internal plate control, the test water was used without addition of the positive control or the test item but with MOPS buffer.- Culture medium different from test medium: no, the holding and breeding of Zebrafish in the laboratories of ibacon are under similar temperature and light conditions. Holding and breeding was performed in Reconstituted Water (ISO Medium, see 6.5). - Intervals of water quality measurement: pH and temperature were determined daily in the freshly prepared and aged test media. At test termination replicates were pooled to obtain sufficient volumes. Total hardness, dissolved oxygen and conductivity were measured at test start and termination only. The appearance of the test item in test water was determined at daily in the freshly prepared and aged test media of all test concentrationsOTHER TEST CONDITIONS- Adjustment of pH: yes with 4-morpholinepropane sulphonic acid (MOPS buffer) 10mM, at pH 6.5 ± 0.2- Photoperiod: The incubation was performed in the dark- Light intensity: 0 luxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Apical Observations: The embryos were observed at test start and after 24, 48, 72 and 96 hours after start of the test for coagulated embryos, lack of somite formation and non-detachment of the tail. The lack of heartbeat was recorded after 48, 72 and 96 hours. All these test parameters were determined using a stereo microscope. Hatching was recorded in all test chambers after 48, 72, and 96 hours.TEST CONCENTRATIONS- Spacing factor for test concentrations: 1.5- Justification for using less concentrations than requested by guideline: /- Range finding study: Pre-experiments were performed under the study number 103971238 in April 2016. The pre-experiments were performed in semi-static test design at pH 6.5, in the dark, with buffered test medium. - Test concentrations: 0.6, 1.4, 3.1, 6.8, 15 mg/L- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (4.0 mg/L)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: coagulation and lack of heartbeat

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

1.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: coagulation and lack of heartbeat

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.912 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: coagulation and lack of heartbeat

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

1.45 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: coagulation and lack of heartbeat

Details on results:

- Behavioural abnormalities: / - Observations on body length and weight:/ - Other biological observations: At 0.415 mg test item/L (mean measured) up to and including 0.912 mg test item/L (mean measured) all fish embryos survived in after 96 hours. At 1.45 mg test item/L (mean measured) 11 embryos showed one of the four apical symptoms (no heartbeat) and in the highest test concentration of 2.12 mg test item/L (mean measured) all fish embryos showed one of the four apical symptoms (no heartbeat or coagulation). At 0.415 mg test item/L (mean measured) hatching success was 75%. At 0.611 mg test item/L (mean measured) hatching success was 85% and at 0.912 mg test item/L (mean measured) hatching success was 95%. At 1.45 mg test item/L (mean measured) hatching success was 45% and at 2.12 mg test item/L (mean measured) no embryo hatched. - Mortality of control: In the negative control one embryo coagulated after 96 hours and in the MOPS buffer control two embryos coagulated. In the internal plate control of 0.415 up to 2.12 mg test item/L all embryos survived and showed 100% hatch after 96 hours. - Other adverse effects control: no, Hatching success in the negative control was 96% and 88% in the MOPS buffer control. - Abnormal responses: / - Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations, all test item solutions were clear throughout the exposure - Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes (mortality should be >30%)In the positive control, containing 4.0 mg dichloroaniline/L, 100% of the embryos died.

Reported statistics and error estimates:

Based on the embryo mortality, the 96-hour LC50 LC20 , LC10 and where possible their 95 %-confidence limits were calculated by probit analysis. The NOEC and the LOEC calculated by Step-down Cochran-Armitage test procedure. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Any other information on results incl. tables

Sublethal observations / clinical signs:

	Test concentrations 96H		Internal plate control 96H
Mean measured conc. (mg test item/L)	% mortality	% hatching	% mortality	% hatching
Negative control	4	96	-	-
MOPS Buffer control	8	88	-	-
Positive control	100	0	0	75
0.42	0	75	0	100
0.61	0	85	0	100
0.91	0	95	0	100
1.45	55	45	0	100
2.12	100	0	0	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The overall fertilisation rate was ≥70%. The temp. was maintained at 26±1 °C. The overall survival of embryos in the negative control was ≥90%. Hatching rate in the negative control was ≥80%. The O2 conc. stayed at ≥80% of saturation.

Conclusions:

Based on the test results the 96-hour LC50 of the test item for zebrafish embryos (Danio rerio) was determined to be 1.44 mg test item/L based on mean measured concentrations. The NOEC was determined to be 0.912 mg test item/L also based on mean measured concentrations.

Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item to fish embryos. For this purpose, Zebrafish embryos were exposed in a semi-static acute fish embryo test to aqueous test media containing the test item at 5 different concentrations according to OECD guideline 236 and GLP. The following endpoints were recorded as indicators of acute lethality in fish embryos: the coagulation of the embryo, lack of somite formation, the non-detachment of the tail-bud from the yolk sac and the lack of heartbeat. Furthermore, the hatching rate was determined. The test item concentrations were verified by HPLC-UV-vis detection. At the start of the test and at the renewal of the test media 86% of the nominal test concentrations were found (average of all test concentrations). After 24 hours test duration, 83% of the nominal value was determined (average of all test concentrations). During the test the fish were exposed to a mean of 85% of nominal. All reported results refer to mean measured concentrations. Based on the test results the 96-hour LC50 of MEXORYL SBU for zebrafish embryos (Danio rerio) was determined to be 1.44 mg test item/L based on mean measured concentrations. The 96 hour NOEC was determined to be 0.912 mg test item/L also based on mean measured concentrations.