(E)-N'-(2-cyano-4-(3-(1-hydroxy-2-methylpropan-2-yl)thioureido)phenyl)-N,N-dimethylformimidamide

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:

Experimental start date 06 January 2020 Experimental completion date 07 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Cas Number:

1429755-57-6

Molecular formula:

C15H12N5OS

IUPAC Name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Specific details on test material used for the study:

Identification: SPS 5290 (Stage 3)
Batch: 80034242B
Purity: 99.5%
Physical State/Appearance: Yellow crystalline powder
Expiry Date: 5 July 2020
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
A sample of each test concentration was taken for immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. Duplicate samples were taken on each occasion and stored frozen for further analysis if required.

Definitive Test
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. An additional sample of each test concentration was incubated alongside the test from which samples were withdrawn for analysis at 24 and 48 hours.
The 0, 24 and 48-Hour samples were stored frozen prior to analysis alongside the 72-Hour test samples. The 72-Hour samples were analyzed on the day of sampling. A set of duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of Elendt M4 media with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following
pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Whatman Polycap filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Whatman Polycap filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
It was evident from these results that the test item was soluble in ASTM culture medium with the aid of prolonged stirring for a period of 24 hours. As a precautionary measure, the preparation was filtered through a 0.2 μm Whatman Polycap filter (approximately 1 liter discarded in order to pre-condition the filter).

Range-Finding Test
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded) to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.4 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL. The stock solutions and each of the prepared concentrations were inverted several times to
ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded) to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 32, 10, 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.8 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Study design

Test type:

static

Water media type:

other: Culture medium

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature wasmaintained at 24 ±1 ºC throughout the test.
The temperature within the incubator was recorded daily.

pH:

The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 8.5 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.

Nominal and measured concentrations:

Range-Finding Test
nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L
Chemical analysis of the test preparations at 0 hours (see Annex 4) showed measured test concentrations to range from 80% to 100% of nominal. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of 42% of nominal at 0.10 mg/L, through to 78% of nominal at 100 mg/L indicating that the test item was unstable under the conditions of the test.

Definitive Test
nominal test concentrations of 100, 32, 10, 3.2, 1.0, 0.32 and 0.10 mg/L.
The geometric mean measured test concentrations were determined to be: 95, 29, 9.5, 2.8, 0.59, 0.41

Details on test conditions:

Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test
The results from the range-finding test showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth rate was observed to be reduced at 100 mg/L. Significant
inhibition of yield was observed at all test concentrations employed with no clear dose response at 0.10, 1.0 and 10 mg/L.
Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.24 x 10^6 cells per mL. Inoculation of 450 mL of test medium with 1.8 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Culture Medium
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Verification of Test Concentrations
Analysis of the 1.0, 3.2, 10, 32 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 86% to 108% of nominal. Analysis of the 0.10 and 0.32 mg/L test preparations showed measured test concentrations of less than the limit of detection (LOD), determined to be 0.026 mg/L, and less than the limit of quantification (LOQ), determined to be 0.82 mg/L respectively. Such results do not indicate that no test item was in solution at these concentrations, just that any test item present was so at a concentration of less than 0.82 mg/L. Measured concentrations in the 3.2, 10, 32 and
100 mg/L test preparations at 24 and 48 and 72 hours ranged from between 72% to 101% of the nominal test concentrations. Analysis of the 1.0 mg/L test preparation at 24 hours showed a measured test concentration of 82% of nominal was obtained, after both 48 and 72 hours the measured concentration had declined to less than the LOQ. Measured concentrations of less than the LOD, or less than the LOQ were obtained from the 0.10 and 0.32 mg/L test preparations throughout.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.41 mg/L) was used to enable calculation of the geometric mean measured concentration. The 0.10 mg/L test group was excluded from the calculation as the measured concentrations obtained throughout were below the LOD or LOQ.

Growth Data
From the data, it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 34 mg/L
ErC20 (0 to 72 hour) : 80 mg/L
ErC50 (0 to 72 hour) : >95 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 24% inhibition of growth rate.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control and the nominal test concentrations of 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L; however, the nominal 100 mg/L test concentration was significantly different (P<0.05). As such the NOEC based on growth rate
was 32 mg/L, equivalent to a geometric mean measured test concentration of 29 mg/L.
Correspondingly the LOEC based on growth rate was 100 mg/L, equivalent to a geometric mean measured test concentration of 95 mg/L.
Inhibition of Yield
EyC10 (0 to 72 hour) : 1.3 mg/L
EyC20 (0 to 72 hour) : 6.5 mg/L
EyC50 (0 to 72 hour) : 55 mg/L
Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05), between the control and the nominal 0.10, 0.32 and 1.0 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05). Therefore the NOEC based on yield was 1.0 mg/L, equivalent to a geometric mean measured test concentration of 0.59 mg/L. Correspondingly the LOEC based on yield was 3.2 mg/L, equivalent to a geometric mean measured test concentration of 2.8 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 177 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 8.87 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the nominal 0.10 and 1.0 mg/L test cultures. Enlarged cells were observed to be present in the control and 0.32 mg/L test cultures. Enlarged cells and cell debris was observed in the 3.2 and 10 mg/L test cultures, whilst misshapen cells were observed to be present in the 32 and 100 mg/L test cultures.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L test cultures were observed to be green dispersions whilst the 100 mg/L test cultures were observed to be extremely pale green dispersions.

Results with reference substance (positive control):

A positive control (Covance Study Number YY61NS) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 20 January 2020 and 06 March 2020.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Growth Rate
EC50 (mg/L): >95*
No Observed Effect Concentration (NOEC) (mg/L): 29
Lowest Observed Effect Concentration (LOEC) (mg/L): 95
Yield
EC50 (mg/L): 55
No Observed Effect Concentration (NOEC) (mg/L): 0.59
Lowest Observed Effect Concentration (LOEC) (mg/L): 2.8
* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 24% inhibition of growth rate.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that the test item was however soluble with the aid of prolonged stirring.

Following a preliminary range-finding test and initial experiment, Raphidocelis subcapitata was exposed to aqueous solutions of the test item at concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The 100 mg/L test concentration was prepared by dissolving the test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded). This test concentration was then further diluted as necessary, to provide the remaining test concentrations.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results.

Analysis of the 1.0, 3.2, 10, 32 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 86% to 108% of nominal. Analysis of the 0.10 and 0.32 mg/L test preparations showed measured test concentrations of less than the limit of detection (LOD), determined to be 0.026 mg/L, and less than the limit of quantification (LOQ), determined to be 0.82 mg/L respectively. Such results do not indicate that no test item was in solution at these concentrations, just that any test item present was so at a concentration of less than 0.82 mg/L. Measured concentrations in the 3.2, 10, 32 and 100 mg/L test preparations at 24 and 48 and 72 hours ranged from between 72% to 101% of the nominal test concentrations. Analysis of the 1.0 mg/L test preparation at 24 hours showed a measured test concentration of 82% of nominal was obtained, after both 48 and 72 hours the measured concentration had declined to less than the LOQ. Measured concentrations of less than the LOD, or less than the LOQ were obtained from the 0.10 and 0.32 mg/L test preparations throughout.

Given the decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations for the 0.32, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations were 0.41, 0.59, 2.8, 9.5, 29 and 95 mg/L respectively.

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Growth Rate

EC50 (mg/L): >95*

No Observed Effect Concentration (NOEC) (mg/L): 29

Lowest Observed Effect Concentration (LOEC) (mg/L): 95

Yield

EC50 (mg/L): 55

No Observed Effect Concentration (NOEC) (mg/L): 0.59

Lowest Observed Effect Concentration (LOEC) (mg/L): 2.8

* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 24% inhibition of growth rate.