Benzonitrile, 4-formyl-3-methoxy-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-13 to 2018-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Test guideline 3

Qualifier:

according to guideline

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-formyl-3-methoxybenzonitrile

EC Number:

804-777-4

Cas Number:

21962-45-8

Molecular formula:

C9 H7 NO2

IUPAC Name:

4-formyl-3-methoxybenzonitrile

Test material form:

solid

Specific details on test material used for the study:

Name: Methoxynitrilaldehyd
IUPAC Name: 4-Formyl-3-methoxybenzonitril
CAS No.: 21962-45-8
Batch No.: BXR8012
Purity: 100.5 % (analytical)
according to certificate of analysis
Certificate of Analysis Date: January 09, 2018
Aggregate State at Room Temperature:solid
Colour: white
Expiry Date: October 24, 2019
Storage Conditions at Test Facility:
At 20 +/- 5 °C, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations 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 concentrations during the test period, duplicate samples from the aged test media of all test concentrations 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 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution of the highest concentration of 100 mg test item/L was prepared by dissolving two times 100 mg test item into 1000 mL test water by shaking for 24 hours. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
Test Concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L (spacing factor 2.2) and a control

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species:Daphnia magna (Straus), clone 5
Age at Test Start: From 2.25 to 19.25 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions:The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

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:

7.9 to 8.1 in the freshly prepared media;
7.8 to 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.7 to 9.0 mg/L in the freshly prepared media;
8.5 to 8.6 mg/L in the aged test media

Nominal and measured concentrations:

nominal: 100, 45.5, 20.7, 9.4 and 4.3 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 dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature:21 °C at test start; 21 °C at test end
pH-Values: .9 to 8.1 in the freshly prepared media; 7.8 to 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.7 to 9.0 mg/L in the freshly prepared media; 8.5 to 8.6 mg/L in the aged test media
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 160 to 390 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.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

30.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

45.5 mg/L

Nominal / measured:

nominal

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 up to and including the test item concentration of 9.4 mg test item/L. At the concentration of 20.7 mg test item/L, one animal was immobile, 19 animals were immobile at the concentration of 45.5 mg test item/L and all 20 animals were immobile at the concentration of 100 mg test item/L.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate performed in January 2018 (study code 88299220), the EC50 after 24 hours was determined to be 1.06 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).
In the reference test performed in June 2017 (study code 88298220) with the reference item potassium dichromate, the EC50 after 24 hours was determined to be 1.58 mg test item/L and the EC50 after 48 hours was determined to be 1.07 mg test item/L.

Reported statistics and error estimates:

Statistical Analysis: The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

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 8.5 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item Methoxynitrilaldehyd to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 20.7 mg test item/L. The 48-hour LOEC was determined to be 45.5 mg test item/L and the 48-hour EC50 value was calculated to be 30.7 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 nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Executive summary:

Title: Methoxynitrilaldehyd: Acute Toxicity to Daphnia magna in a Semi-Static 48-hour Immobilisation Test

Purpose:

The purpose of this study was to determine the influence of the test item Methoxynitrilaldehyd on the mobility of Daphnia magna.

For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test to various concentrations 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: Methoxynitrilaldehyd; batch no.: BXR8012; purity: 100.5 % (analytical) according to certificate of analysis

Test Species: Female Daphnia magna, clone 5; 2.25 to 19.25 hours old

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

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a 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 Concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L (spacing factor 2.2) and a control

Test Conditions: Water temperature: 20.5 to 21.4 °C; pH value: 7.8 to 8.1; dissolved oxygen concentration: 8.5 to 9.0 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 260 to 390 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 up to and including the test item concentration of 9.4 mg test item/L. At the concentration of 20.7 mg test item/L, one animal was immobile, 19 animals were immobile at the concentration of 45.5 mg test item/L and all 20 animals were immobile at the concentration of 100 mg test item/L.

Analytical test results:

 The quantification of the test item Methoxynitrilaldehyd in the test samples was performed using liquid chromatography with UV detection.

In the freshly prepared test media at the start of the test and at the renewal of the test media 107 % of the nominal test concentrations were found (average of all test concentrations). In the aged test media after 24 hours test duration and at test end, 100 % of the nominal value was determined (average of all test concentrations). During the test the test organism were exposed to a mean of 103 % of nominal.

 

Conclusion: The toxic effect of the test item Methoxynitrilaldehyd to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 20.7 mg test item/L. The 48-hour LOEC was determined to be 45.5 mg test item/L and the 48-hour EC50 value was calculated to be 30.7 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 nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test. This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.