2-Propenal, 2-fluoro-3-(4-morpholinyl)-, (Z)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-11-01 to 2019-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study; EU method; GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

July 28, 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2Z)-2-fluoro-3-(morpholin-4-yl)prop-2-enal

EC Number:

813-789-9

Cas Number:

152873-67-1

Molecular formula:

C7H10FNO2

IUPAC Name:

(2Z)-2-fluoro-3-(morpholin-4-yl)prop-2-enal

Test material form:

solid

Specific details on test material used for the study:

Name: Fluormorpholinylacrylaldehyd (FMA)
Batch No.: BXR82PT
Purity: 86.7 % according to the certificate of analysis (from January 26, 2018), given in the Study Plan
Purity: 98.6 % according to the certificate of analysis (after reanalysis of the test item)
Certificate of Analysis Date: September 04, 2019 (reanalysis of the test item);
January 26, 2018 (given in the Study Plan)
Aggregate State at Room Temperature: Solid
Colour: Orange-brownish
Expiry Date: August 21, 2019 after reanalysis
(November 01, 2018 according to the certificate of analysis from January 26, 2018 given in the Study Plan)
Storage Conditions at the Test Facility: After receipt at the test facility for 7 days at 4 ± 4 °C until storage recommendations were provided; thereafter at 20 +/- 5 °C in the dark.

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
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 test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control. Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.9 mg test item into 1009 mL test water by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: Coloration of the test medium of the highest test item concentration at test start and after 24 h caused by the test item.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 °C

pH:

7.8 - 8.0 at test start and 8.9 to 9.6 at test end

Nominal and measured concentrations:

100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control.

Details on test conditions:

Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.6 to 22.3 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test. pH was: 7.8 to 8.0 at test start and 8.9 to 9.6 at test end
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5540 lux (range: 5310 to 5960 lux)
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The 72-hour EyC50 was calculated to be > 100 mg test item/L and the ErC50 > 100 mg test item/L. The 72-hour EyC10 was calculated to be 13.4 mg test item/L and the ErC10 > 100 mg test item/L. The 72-hour NOEyC was determined to be 10 mg test item/L and the associated 72-hour LOEyC of 32 mg test item/L. The 72-hour NOErC was determined to be 10 mg test item/L and the associated 72-hour LOErC is 32 mg test item/L.
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in June 2018
Parameter (0-72 h)
72-hour EC50
Yield [mg test item/L]: 0.442
Growth rate [mg test item/L]: 1.11
Biomass [mg test item/L]: 0.487
72-hour NOEC
Yield [mg test item/L]/Growth rate [mg test item/L]/Biomass [mg test item/L]: 0.094
72-hour LOEC
Yield [mg test item/L]/Growth rate [mg test item/L]/Biomass [mg test item/L]:0.21
values refer to nominal test concentrations

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
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:

, Cell Density Increase: 203.2-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 9.5 %; Coefficient of Variation of Average Growth: 1.5 %

Conclusions:

The influence of Fluormorpholinylacrylaldehyd (FMA) on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was determined to be > 100 mg test item/L, and the 72-hour ErC50 value was not determinable. The 72-hour NOEyC and NOErC were determined to be 10 mg test item/L and the associated 72-hour LOEyC and LOErC were determined to be 32 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:

Fluormorpholinylacrylaldehyd (FMA): Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose:

The purpose of this test was to determine the inhibitory effect of the test item Fluormorpholinylacrylaldehyd (FMA) on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours hours, and thus over several algal generations. The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of 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/Recommendations:

-OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

-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: Fluormorpholinylacrylaldehyd (FMA); Batch No.: BXR82PT; purity: 98.6 %, according to certificate of analysis.

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrically measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control.

Test Conditions:

Water temperature: 21.6 to 22.3 °C; pH values at test start 7.8 to 8.0, at the end of the test 8.9 to 9.6; continuous illumination; mean light intensity: 5540 lux (5310 to 5960 lux).

Results

Biological Results:

The 72-hour EyC50 was calculated to be > 100 mg test item/L and the  ErC50 > 100 mg test item/L. The 72-hour EyC10 was calculated to be 13.4 mg test item/L and the ErC10 > 100 mg test item/L. The 72-hour NOEyC was determined to be 10 mg test item/L and the associated 72-hour LOEyC of 32 mg test item/L. The 72-hour NOErC was determined to be 10 mg test item/L and the associated 72-hour LOErC is 32 mg test item/L.

The microscopic examination of the shape of the algal cells after  72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Analytical Results:

The quantification of the test item Fluormorpholinylacrylaldehyd (FMA) in the test samples was performed using liquid chromatography with UV detection.

At the start of the test 98% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 96 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 97 % of nominal.

Conclusion:

The influence of Fluormorpholinylacrylaldehyd (FMA) on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was determined to be > 100 mg test item/L, and the 72-hour ErC50 value was not determinable. The 72-hour NOEyC and NOErC were determined to be 10 mg test item/L and the associated 72-hour LOEyC and LOErC were determined to be 32 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 acceptable and satisfies the guideline requirements for growth inhibition algae studies.