2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-09-2016 to 14-10-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

dated August 24, 2009

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

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.
Duplicate samples were taken from all freshly prepared test media (without algae) of all test concentrations at the start and at the end of the test to exclude any effect of the algae on the analysis.
Additional samples of the control blank were taken at test end without any sample treatment.

Vehicle:

no

Details on test solutions:

Pre-Experiments: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
The pre-experiments were not performed in compliance with the GLP-Regulations and are be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.

Test Concentrations: Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested.
Corresponding to following mean measured concentrations of the test item and/or it´s degradation products:
45.5, 11.9, 6.47, 2.25 and 2.25 mg/L, and a control.

Control: In the control, test water was used without addition of the test item.

Dosage of Test Item: A defined amount of the test item was added directly to the test water for the three highest test concentration and was carefully stirred for 48 hours in the dark to dissolve as much of the test item as possible. The highest test item concentration of 100 mg test item/L was prepared by mixing 200 mg test item into 2000 mL test water, for the test item concentration of 32 mg test item/L, 64.4 mg test item into 2012.5 mL test water and for the concentration of 10 mg test item/L, 20.4 mg test item into 2040 mL test water. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium. The test concentrations of nominal 3.2 and 1.0 mg test item/L were diluted from the final test solution of nominal 10 mg test item/L.
The test media were prepared just before introduction of the algae (= start of the test).

Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms (species):

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

Details on test organisms:

Test System
Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

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 to 23 °C

pH:

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: 7.7 to 8.0 at test start and 8.6 to 9.9 at test end
The pH in the control increased by more than 1.5 units in the control medium. However, this does not invalidate the tests since the validity criterion was met.

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of
100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested.
Corresponding to following mean measured concentrations of the test item and/or it´s degradation products:
45.5, 11.9, 6.47, 2.25 and 2.25 mg/L, and a control.

Details on test conditions:

Course of the Test
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.

Replicates: The test was performed with three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.

Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item (see also "Determination of the Cell Density").

Exposure Time: 72 hours

Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

Endpoints: Yield and growth rate of the algae

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 45.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Biological Results

Parameter Yield [mg test item/L] Growth rate [mg test item/L]

72-hour EC50 19.5 > 45.5
95 % conf. interval 17.4 - 22.2 n.d.

72-hour EC20 9.37 29.0 95 % conf. interval 7.96 - 10.7 26.8 - 31.1

72-hour EC10 6.39 17.0
95 % conf. interval 5.13 - 7.56 14.7 - 19.0

72-hour NOEC 2.25 6.47

72-hour LOEC 6.47 11.9

n.d. = not determinable
Values refer to mean measured test concentrations


Microscopic Examination:
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 32 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 second highest concentration tested. In the highest test concentration of nominal 100 mg test item/L, cells were observed to be larger than in the control.

Results with reference substance (positive control):

Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in February 2016 (Project No.: 110663210)

Parameter Yield Growth rate Biomass
(0-72 h) [mg test item/L] [mg test item/L] [mg test item/L]

72-hour EC50 0.721 1.50 0.794
95 % conf. interval 0.588 - 0.847 1.43 - 1.58 0.668 - 0.910

72-hour EC20 0.531 0.731 0.552
95 % conf. interval 0.367 - 0.638 0.673 - 0.785 0.381 - 0.658

72-hour EC10 0.452 0.502 0.456
95 % conf. interval 0.280 - 0.562 0.447 - 0.554 0.278 - 0.569

72-hour NOEC 0.45 0.45 0.45
72-hour LOEC 1.0 1.0 1.0

Values refer to nominal test concentrations

Limit of Detection:

2.6 mg TC/L

1.3 mg IC/L

Limit of Detections were calculated according to DIN 32645.

Limit of Quantification:

4 mg TOC/L

90 % (n = 5, RSD 1%, see Table 14)

Mean Recovery in the Test Samples:

Freshly prepared: 46 % (n = 6, RSD 21 %)

Aged test media: 52 % (n = 6, RSD 31 %)

 

Summary of Analytical Results

sample description       % of              RSD                      Mean measured concentration

[mg test item/L]     nominal(1)           [%]            n              [mg test item/L]

Control                  n.a.                      n.a.           4                  n.a.

1.0                      n.a.                        n.a.            4                 2.25

3.2                       63                        29             4                 2.25

10                        65                        14              4                6.47

32                        37                         2              4                11.9

100                     46                           1              4                45.5

(1) mean value of all measured samples per treatment group

italic: Values below the LOQ. For Values <LOQ, half the LOQ was used for

calculating measured concentrations according to Guideline OECD No.23. RSD: relative standard

deviation per treatment group

n: number of analysed samples

n.a.: not applicable

At the start of the test 46 % of the nominal test concentrations

Validity criteria fulfilled:

yes

Conclusions:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 19.5 mg test item/L, and the 72-hour ErC50 value was calculated to be > 45.5 mg test item/L.
The 72-hour NOEyC was determined to be 2.25 mg test item/L and the associated 72-hour LOEyC was 6.47 mg test item/L.
The 72-hour NOErC was determined to be 6.47 mg test item/L and the associated 72-hour LOErC was 11.9 mg test item/L.

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

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, and thus over several algal generations.

The method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.

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 concentrations were inoculated with 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 spectrophotometrical measurement.

The Endpoints are Yield and growth rate of the algae

Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested. Corresponding to following mean measured concentrations of the test item and/or it´s degradation products:

45.5, 11.9, 6.47, 2.25 and 2.25 mg/L, and a control.

The quantification of the test item and / or its degradation products was performed by analysing the total organic carbon (TOC).

At the start of the test 46 % of the nominal test concentrations were found (average of the nominal concentrations of 100, 32, and 10 mg test item/L). After 72 hours test duration, 52 % of the nominal value was determined (average of the nominal concentrations of 100, 32, and 10 mg test item/L). During the test

the algae were exposed to a mean of 49 % of nominal. All reported results refer to mean measured concentrations.

Conclusion:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 19.5 mg test item/L, and the 72-hour ErC50 value was calculated to be > 45.5 mg test item/L.

The 72-hour NOEyC was determined to be 2.25 mg test item/L and the associated 72-hour LOEyC was 6.47 mg test item/L.

The 72-hour NOErC was determined to be 6.47 mg test item/L and the associated 72-hour LOErC was 11.9 mg test item/L.

Description of key information

The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

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, and thus over several algal generations.

The method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.

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 concentrations were inoculated with 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 spectrophotometrical measurement.

The Endpoints are Yield and growth rate of the algae

Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested. Corresponding to following mean measured concentrations of the test item and/or it´s degradation products:

45.5, 11.9, 6.47, 2.25 and 2.25 mg/L, and a control.

The quantification of the test item and / or its degradation products was performed by analysing the total organic carbon (TOC).

At the start of the test 46 % of the nominal test concentrations were found (average of the nominal concentrations of 100, 32, and 10 mg test item/L). After 72 hours test duration, 52 % of the nominal value was determined (average of the nominal concentrations of 100, 32, and 10 mg test item/L). During the test

the algae were exposed to a mean of 49 % of nominal. All reported results refer to mean measured concentrations.

Conclusion:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 19.5 mg test item/L, and the 72-hour ErC50 value was calculated to be > 45.5 mg test item/L.

The 72-hour NOEyC was determined to be 2.25 mg test item/L and the associated 72-hour LOEyC was 6.47 mg test item/L.

The 72-hour NOErC was determined to be 6.47 mg test item/L and the associated 72-hour LOErC was 11.9 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

45.5 mg/L

EC10 or NOEC for freshwater algae:

6.47 mg/L