2-pentylcyclopentan-1-one

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across approach is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological, ecotoxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances (Table 1) are 1,2-branched pentyl cyclopentane. The source substance is an alcool, while the target substance is a ketone. The source substance is the secondary alcohol formed by metabolism of the Target substance.

3. ANALOGUE APPROACH JUSTIFICATION
Based on the close structural similarity, on the similar basic physico-chemical properties and the same environmental profile, both target and source substances are not expected to have significantly difference on the aquatic toxicity.
The study performed on the source substance (OECD 203, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the toxicity to aquatic algae and cyanobacteria study conducted with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement.

4. DATA MATRIX
See Iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 11.3-17.5 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.8-6.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See table 6.1.5/3 in "Any other information on results incl. tables".
The test item had a significant inhibitory effect on the yield and growth rate of the algae after the test period of 72 hours at the mean measured concentration of 6.6 (nominal 10 mg/L) and at all higher test concentrations.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.20 mg/L

Reported statistics and error estimates:

None

Table 6.1.5/1: Results obtained for the concentrations of the treatment samples

Age (day)	Nominal concentration of the test item (mg/L)	Measured concentration of test item (mg/L)			Sample preparation factor	Determined average concentration of test item (mg/L)	Recovery rate (%)
		Sample 1	Sample 2	Average
0	Control 0.32 1 3.2 10 32 100	<LOQana 0.234 0.756 2.94 7.73 21.5 104	<LOQana 0.167 0.836 2.92 7.44 23.0 108	n.a. 0.200 0.796 2.93 7.59 22.3 106	1.01 1.01 1.01 1.01 1.01 1.01 1.01	n.a. 0.202 0.804 2.96 7.66 22.5 107	n.a. 63 80 92 77 70 107
3	Control 0.32 1 3.2 10 32 100	<LOQana <LOQana <LOQana 0.990 5.33 22.6 92.7	<LOQana <LOQana <LOQana 0.985 5.78 20.8 94.4	n.a. n.a. n.a. 0.988 5.55 21.7 93.5	1.01 1.01 1.01 1.01 1.01 1.01 1.01	n.a. n.a. n.a. 0.998 5.61 21.9 94.5	n.a. n.a. n.a. 31 56 68 94

LOQana = 0.104 mg test item/L

n.a. = not applicable

Table 6.1.5/2: Mean measured test item concentrations

Nominal test concentration (mg/L)	Mean measured concentration of the test item (geometric mean)
	(mg/L)	(% of nominal)
Control 0.32 1 3.2 10 32 100	- 0.10* 0.21* 1.7 6.6 22 101	- 32 21 54 66 69 101

* The measured concentration at the end of the test was below the limit of detection (LOQ = 0.104 mg/L). The mean measured concentration is based on the measured concentration from the start of the test and half of the LOQ (= 0.0525 mg/L) for test end.

Table 6.1.5/3: Biomass of algae

Nominal concentration (mg/L)	Measured concentration (mg/L)	Repl. No.	Biomass of algae* (relative fluorescence units)			Inhibition of growth rate (%)
			24h	48h	72h
Control	-	1 2 3 4 5 6	5.0 5.1 4.8 4.6 5.5 5.1	34.4 33.8 31.2 39.7 39.8 42.6	129.3 142.1 156.9 175.4 171.6 138.6	0.0
		Mean SD	5.0 0.3	36.9 4.4	152.3 18.7
0.32	0.10	1 2 3	4.3 5.1 4.9	29.9 35.2 34.1	187.5 186.6 181.0	-2.7
		Mean SD	4.8 0.4	33.1 2.8	185.0 3.5
1.0	0.21	1 2 3	4.7 3.7 3.6**	31.6 30.5 3.9**	153.3 182.8 1.3**	-1.9
		Mean SD	4.2 0.8	31.0 0.8	168.0 20.8
3.2	1.7	1 2 3	4.5 4.3 4.7	14.1 22.8 24.0	53.4 135.5 157.8	6.9
		Mean SD	4.5 0.2	20.3 5.4	115.6 55.0
10	6.6	1 2 3	4.7 3.6 3.8**	17.3 12.3 4.0**	103.0 43.9 0.9**	15.1
		Mean SD	4.1 0.8	14.8 3.5	73.5 41.7
32	22	1 2 3	2.6 3.7 3.1	3.1 4.9 3.7	2.8 3.2 3.4	72.3
		Mean SD	3.2 0.5	3.9 0.9	3.1 0.3
100	101	1 2 3	1.4 1.4 1.3	1.1 1.1 1.4	0.3 0.5 0.5	108.6
		Mean SD	1.3 0.1	1.2 0.2	0.4 0.1

SD: Standard deviation.

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10^3). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 0.7 x 10^3 relative fluorescence units).

**: Replicate no. 3 of the nominal concentrations of 1.0 and 10 mg/L, respectively, was considered as outlier (no algal growth) and was not taken into account for the evaluation.

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate, the 72h-ErC10 and 72h-ErC50 were determined to be 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L) and 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L), respectively. The 72h-NOECr was 1.7 mg/L.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72h static test according to the EU Method C.3 and the OECD Guideline 201 with GLP statement.

The nominal concentrations of the test item of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control. Since the test item was determined to be volatile, the test was performed in a closed system to avoid losses of the volatile substance.

The measured concentrations of the test item in the test media samples were between 63 and 107% of nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred at least in the lower test concentrations. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 21 and 101% of nominal values.

The biological results were related to the mean measured test item concentrations, which were 0.10, 0.21, 1.7, 6.6, 22 and 101 mg/L, respectively, corresponding to the nominal test item concentrations mentioned above. Unfortunately, the geometric spacing of critical measured concentrations between 15 and 72% effects was greater than a factor of 3.2 as recommended in the guideline due to substance loss at the lower concentration.

Based on the growth rate, the 72h-ErC10 and 72h-ErC50 were determined to be 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L) and 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L), respectively. The 72h-NOEC was 1.7 mg/L.

All validity criteria were fulfilled. In the control, the biomass increased by a factor of 214 over 72 hours. The mean coefficient of variation of the daily growth rates in the control (section by section growth rates) during 72 hours was 18.4% and the coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 2.3%.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 August to 03 September 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed according to the EU Method C.3 and the OECD Guideline 201 with GLP statement. All validity criteria were fulfilled and no deviations were observed. However, the geometric spacing of critical measured concentrations between 15 and 72% effects was greater than a factor of 3.2 as recommended in the guideline due to substance loss at the lower concentration. In addition, validation applies with restrictions as the test substance is not the registered substance. Read-across is justified as data relates to a chemical substance structurally similar to the registered substance.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP compliance inspected on 05-09 and 26-30 November 2007 / signed on 12 November 2008

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.
For sampling at the end of the test, the test medium of the treatment replicates was pooled. The samples were analysed immediately after sampling.
The concentrations of the test item were determined in the all duplicate test medium samples. From the control samples, one of the duplicate samples was analysed from the corresponding sampling times.
The samples (2 mL) were directly pipetted into headspace vials containing 0.6 g sodium chloride and further 20 µL acetone were added (giving a dilution factor of 1.01). Subsequently, the samples were analysed by Headspace-GC/FID without further storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.6 mg of the test item completely in 1000 mL of test water using intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Controls: one control without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen / Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the test.
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

24 mg/L as CaCO3.

Test temperature:

The water temperature during the test was between 23 and 24°C.

pH:

At the start of the test, the pH measured in the treatments was 8.2. At the end of the test, pH values of 8.2 to 8.8 were measured. The increase of the pH during the test was caused by the uptake of CO2 by algae due to their growth despite the test media were stirred during the test.

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Measured concentrations: See tables 6.1.5/1 and 6.1.5/2 in "Any other information on results incl. tables".
The measured concentrations of the test item in the test media samples were between 63 and 107% of nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred at least in the lower test concentrations. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 21 and 101% of nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: completely filled with test solution and tightly closed with a glass stopper.
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 10000 cells/mL.
- Control end cells density: See table 6.1.5/3 in "Any other information on results incl. tables".
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines, modified according to the International Standard ISO 14442, was used for algal cultivation and testing.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: Fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. Approx. 7000 Lux (range 6500 to 7500 Lux, measured in nine places in the experimental area).
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800).
- Chlorophyll measurement: no
- Other: At the end of the test, a sample was taken from the control and from the test concentration of nominal 100 mg/L. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes.
- Test concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 11.3-17.5 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.8-6.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See table 6.1.5/3 in "Any other information on results incl. tables".
The test item had a significant inhibitory effect on the yield and growth rate of the algae after the test period of 72 hours at the mean measured concentration of 6.6 (nominal 10 mg/L) and at all higher test concentrations.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.20 mg/L

Reported statistics and error estimates:

None

Table 6.1.5/1: Results obtained for the concentrations of the treatment samples

Age (day)	Nominal concentration of the test item (mg/L)	Measured concentration of test item (mg/L)			Sample preparation factor	Determined average concentration of test item (mg/L)	Recovery rate (%)
		Sample 1	Sample 2	Average
0	Control 0.32 1 3.2 10 32 100	<LOQana 0.234 0.756 2.94 7.73 21.5 104	<LOQana 0.167 0.836 2.92 7.44 23.0 108	n.a. 0.200 0.796 2.93 7.59 22.3 106	1.01 1.01 1.01 1.01 1.01 1.01 1.01	n.a. 0.202 0.804 2.96 7.66 22.5 107	n.a. 63 80 92 77 70 107
3	Control 0.32 1 3.2 10 32 100	<LOQana <LOQana <LOQana 0.990 5.33 22.6 92.7	<LOQana <LOQana <LOQana 0.985 5.78 20.8 94.4	n.a. n.a. n.a. 0.988 5.55 21.7 93.5	1.01 1.01 1.01 1.01 1.01 1.01 1.01	n.a. n.a. n.a. 0.998 5.61 21.9 94.5	n.a. n.a. n.a. 31 56 68 94

LOQana = 0.104 mg test item/L

n.a. = not applicable

Table 6.1.5/2: Mean measured test item concentrations

Nominal test concentration (mg/L)	Mean measured concentration of the test item (geometric mean)
	(mg/L)	(% of nominal)
Control 0.32 1 3.2 10 32 100	- 0.10* 0.21* 1.7 6.6 22 101	- 32 21 54 66 69 101

* The measured concentration at the end of the test was below the limit of detection (LOQ = 0.104 mg/L). The mean measured concentration is based on the measured concentration from the start of the test and half of the LOQ (= 0.0525 mg/L) for test end.

Table 6.1.5/3: Biomass of algae

Nominal concentration (mg/L)	Measured concentration (mg/L)	Repl. No.	Biomass of algae* (relative fluorescence units)			Inhibition of growth rate (%)
			24h	48h	72h
Control	-	1 2 3 4 5 6	5.0 5.1 4.8 4.6 5.5 5.1	34.4 33.8 31.2 39.7 39.8 42.6	129.3 142.1 156.9 175.4 171.6 138.6	0.0
		Mean SD	5.0 0.3	36.9 4.4	152.3 18.7
0.32	0.10	1 2 3	4.3 5.1 4.9	29.9 35.2 34.1	187.5 186.6 181.0	-2.7
		Mean SD	4.8 0.4	33.1 2.8	185.0 3.5
1.0	0.21	1 2 3	4.7 3.7 3.6**	31.6 30.5 3.9**	153.3 182.8 1.3**	-1.9
		Mean SD	4.2 0.8	31.0 0.8	168.0 20.8
3.2	1.7	1 2 3	4.5 4.3 4.7	14.1 22.8 24.0	53.4 135.5 157.8	6.9
		Mean SD	4.5 0.2	20.3 5.4	115.6 55.0
10	6.6	1 2 3	4.7 3.6 3.8**	17.3 12.3 4.0**	103.0 43.9 0.9**	15.1
		Mean SD	4.1 0.8	14.8 3.5	73.5 41.7
32	22	1 2 3	2.6 3.7 3.1	3.1 4.9 3.7	2.8 3.2 3.4	72.3
		Mean SD	3.2 0.5	3.9 0.9	3.1 0.3
100	101	1 2 3	1.4 1.4 1.3	1.1 1.1 1.4	0.3 0.5 0.5	108.6
		Mean SD	1.3 0.1	1.2 0.2	0.4 0.1

SD: Standard deviation.

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10^3). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 0.7 x 10^3 relative fluorescence units).

**: Replicate no. 3 of the nominal concentrations of 1.0 and 10 mg/L, respectively, was considered as outlier (no algal growth) and was not taken into account for the evaluation.

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate, the 72h-ErC10 and 72h-ErC50 were determined to be 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L) and 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L), respectively. The 72h-NOECr was 1.7 mg/L.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72h static test according to the EU Method C.3 and the OECD Guideline 201 with GLP statement.

The nominal concentrations of the test item of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control. Since the test item was determined to be volatile, the test was performed in a closed system to avoid losses of the volatile substance.

The measured concentrations of the test item in the test media samples were between 63 and 107% of nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred at least in the lower test concentrations. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 21 and 101% of nominal values.

The biological results were related to the mean measured test item concentrations, which were 0.10, 0.21, 1.7, 6.6, 22 and 101 mg/L, respectively, corresponding to the nominal test item concentrations mentioned above. Unfortunately, the geometric spacing of critical measured concentrations between 15 and 72% effects was greater than a factor of 3.2 as recommended in the guideline due to substance loss at the lower concentration.

Based on the growth rate, the 72h-ErC10 and 72h-ErC50 were determined to be 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L) and 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L), respectively. The 72h-NOEC was 1.7 mg/L.

All validity criteria were fulfilled. In the control, the biomass increased by a factor of 214 over 72 hours. The mean coefficient of variation of the daily growth rates in the control (section by section growth rates) during 72 hours was 18.4% and the coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 2.3%.

Description of key information

Read-across, EU Method C.3, OECD Guideline 201, GLP, key study, validity 2:
72h-ErC50 = 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L).
72h-ErC10 = 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L).
72h-NOECr = 1.7 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

14 mg/L

EC10 or NOEC for freshwater algae:

1.7 mg/L

Additional information

To assess the toxicity of the registered substance, 2 -pentylcyclopentan-1-one, to aquatic algae, one valid experimental study is available.

 

This GLP study, assessed as a key study, was performed on a read-across substance, 2 -pentylcyclopentan-1-ol, according to EU Method C.3 and OECD Guideline 201. Both compounds (the registered substance and the read-across substance) acting with a non-polar narcotic MOA (Class 1) and are structurally related, in that each has an alkyl chain with five carbon (pentyl) with a cyclopentane ring. Both compounds contain a 2 -pentylcyclopentane skeleton. The cyclopentane ring is associated with a ketone functional group for the registered substance and with an alcohol functional group for the read-across substance.

In this study, the algae Pseudokirchneriella subcapitata were exposed to the test substance (nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L) in a static condition during 72 hours, to assess the influence of the test substance on the growth of the freshwater green algal species. Additionally, a control group was tested in parallel. Since the test substance was determined to be volatile, the test was performed in a closed system to avoid losses of the volatile substance. The measured concentrations of the test substance in the test media samples were between 63 and 107% of nominal values at the start of the test. During the test period of 72 hours, a decrease of test substance concentration in the test media occurred at least in the lower test concentrations. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 21 and 101% of nominal values. The biological results were related to the mean measured test item concentrations, which were 0.10, 0.21, 1.7, 6.6, 22 and 101 mg/L, respectively, corresponding to the nominal test item concentrations mentioned above. Unfortunately, the geometric spacing of critical measured concentrations between 15 and 72% effects was greater than a factor of 3.2 as recommended in the guideline due to substance loss at the lower concentration. Based on the growth rate, the 72h-ErC10 and 72h-ErC50 were determined to be 4.6 mg/L (95% confidence interval: 2.8 -6.2 mg/L) and 14.0 mg/L (95% confidence interval: 11.3 -17.5 mg/L), respectively. The 72h-NOEC was 1.7 mg/L.

This supporting substance is considered adequate for read-across purposes based on structural similarity, on similar basic physico-chemical properties and similar environmental profile as well as the same mode of action between the registered substance and the analogue material used. In addition, as the log Kow value of the analogue material is slightly greater than the registered substance, the read-across approach is considered as a worst case scenario (see IUCLID section 13 for justification).