Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate

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:

Experimental start date: 25 May 2017. Experimental completion date: 17 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Lyrame
Physical state/Appearance: Yellow viscous liquid
Storage Conditions: Approximately 4 ºC in the dark under nitrogen

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Preliminary investigational work showed that increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.

Range finding test
Nominal amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (8.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

Definitive test
Nominal amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 liters of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded). After siphoning the aqueous phase was passed through one sheet of filter paper to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

ACCLIMATION
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.0 at 0 hours to pH 7.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Range finding test: The range-finding test was conducted to nominal loading rates of 10 and 100 mg/L.
Definitive Test: Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
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 material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.30 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 7.6 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.

- No. of vessels per concentration (replicates):
3

- No. of vessels per control (replicates):
6

- No. of vessels per vehicle control (replicates):
None

GROWTH MEDIUM
- Standard medium used: yes
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The culture medium was prepared using reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

- Photoperiod:
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

- Light intensity and quality:
intensity approximately 7000 lux

- Salinity (for marine algae):
Not applicable

- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 20, 44 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

- Results used to determine the conditions for the definitive study:
The results showed no significant effect on growth rate at 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF.
Based on this information loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.11 mg/L, to 0.22 mg/L. Measured test concentrations of less than the LOQ were obtained from all test preparations at 72 hours.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella 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:

Inhibition of growth rate
ErL10 (0 - 72 h) : 21 mg/L loading rate WAF
ErL20 (0 - 72 h) : 31 mg/L loading rate WAF
ErL50 (0 - 72 h) : 58 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all loading rates 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 between the control, 6.25, 12.5 and 25 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 25 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 50 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h) : 22 mg/L loading rate WAF
EyL20 (0 - 72 h) : 26 mg/L loading rate WAF
EyL50 (0 - 72 h) : 33 mg/L loading rate WAF; 95% confidence limits 30 - 35 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.3.3. There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 25 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 50 mg/L loading rate WAF.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 143 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 : 7.17 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% 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 – 72 h) was 1% 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 control or test cultures at 6.25, 12.5 and 25 mg/L loading rate WAF, however no viable cells was observed to be present in the test cultures at 50 and 100 mg/L loading rate WAF.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of mixing all loading rate WAFs were observed to have formed clear colorless media columns with test item adhered to the glass slide suspended within the media column. After stirring, and following a 1-Hour standing period the 6.25, 12.5, 25 and 50 mg/L loading rate WAFs were observed to have formed clear colorless media columns with test item adhered to the glass slides suspended within the media column. The 100 mg/L loading rate WAF was observed to have formed a very slightly hazy media column with test item adhered to the glass slide suspended within the media column, some globules of test item were observed to be settled on the bottom of the mixing vessel. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the media column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug. After siphoning the aqueous phase was passed through one sheet of filter paper to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.
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, 6.25, 12.5 and 25 mg/L loading rate WAFs were observed to be green dispersions whilst the 50 and 100 mg/L loading rate WAFs were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Dissiminated (delete when editing): Fill e.g. copy paste from test report when applicable may be important when deriving EC10s and reliability of the statistics.

Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.60E+03	6.20E+05	-	-
	R2	5.72E+03	5.66E+05
	Mean	5.66E+03	5.93E+05
10	R1	5.60E+03	5.36E+05	5	16
	R2	5.60E+03	4.66E+05
	Mean	5.60E+03	5.01E+05
100	R1	5.02E+03	2.39E+04	72	98
	R2	5.90E+03	1.57E+04
	Mean	5.46E+03	1.98E+04

*       Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)		pH	Cell Densities*(cells per mL)			pH
		0 h	20 h	44 h	72 h	72 h
Control	R1	7.0	1.92E+04	1.09E+05	7.86E+05	7.2
	R2		1.74E+04	1.03E+05	7.09E+05
	R3		1.76E+04	1.17E+05	7.18E+05
	R4		2.01E+04	1.35E+05	7.86E+05
	R5		1.89E+04	1.21E+05	6.87E+05
	R6		1.84E+04	1.02E+05	6.12E+05
	Mean		1.86E+04	1.14E+05	7.17E+05
6.25	R1	7.2	1.94E+04	1.03E+05	6.83E+05	7.3
	R2		1.72E+04	8.64E+04	6.24E+05
	R3		1.52E+04	8.19E+04	5.63E+05
	Mean		1.72E+04	9.04E+04	6.23E+05
12.5	R1	7.1	1.82E+04	1.09E+05	7.06E+05	7.3
	R2		1.72E+04	8.64E+04	5.83E+05
	R3		1.78E+04	9.13E+04	5.85E+05
	Mean		1.77E+04	9.56E+04	6.25E+05
25	R1	7.2	1.73E+04	1.13E+05	5.39E+05	7.3
	R2		1.65E+04	1.16E+05	5.37E+05
	R3		1.77E+04	9.80E+04	7.15E+05
	Mean		1.72E+04	1.09E+05	5.97E+05
50	R1	7.1	7.17E+03	1.62E+04	5.75E+04	7.3
	R2		5.33E+03	1.36E+04	5.85E+04
	R3		5.74E+03	1.72E+04	5.54E+04
	Mean		6.08E+03	1.57E+04	5.71E+04
100	R1	7.4	8.16E+03	1.14E+04	2.84E+04	7.3
	R2		6.37E+03	9.54E+03	1.78E+04
	R3		6.53E+03	1.17E+04	1.87E+04
	Mean		7.02E+03	1.09E+04	2.16E+04

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Validity criteria fulfilled:

yes

Conclusions:

The ErL50, ErL10 and NOELR were 58, 21 and 25 mg/l

Executive summary:

A study was performed to assess the effect of the test material, Lyrame on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Test solutions were prepared by taking nominal amounts of test item (12.5, 25, 50, 100 and 200 mg) each separately weighed onto a glass slide and suspended within 2 liters of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded). After siphoning the aqueous phase was passed through one sheet of filter paper to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAF.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.11 mg/L, to 0.22 mg/L. Measured test concentrations of less than the LOQ were obtained from all test preparations at 72 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results, EL50: 58 mg/L, NOELR: 25 mg/L, LOELR: 50 mg/L.

Description of key information

A study was performed to assess the effect of the test material, Lyrame on the growth of the green alga Pseudokirchneriella subcapitata.The method followed that described in the OECD TG No 201. Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Test solutions were prepared by taking nominal amounts of test item (12.5, 25, 50, 100 and 200 mg) each separately weighed onto a glass slide and suspended within 2 liters of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded). After siphoning the aqueous phase was passed through one sheet of filter paper to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAF.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.11 mg/L, to 0.22 mg/L. Measured test concentrations of less than the LOQ were obtained from all test preparations at 72 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results, EL50: 58 mg/L, NOELR: 25 mg/L, LOELR: 50 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

58 mg/L

EC10 or NOEC for freshwater algae:

21 mg/L