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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - October 2014
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
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes
Specific details on test material used for the study:
Description: Yellow viscous liquid
Batch: SC00010629
Purity/Composition: UVCB substance
Analytical monitoring:
yes
Details on sampling:
Samples for TOC analysis were taken at test initiation and after 72 hours.
Vehicle:
no
Details on test solutions:
The test item was prepared as a Water Accommodated Fraction (WAF). Weighing of test substance was performed under either dimmed light (combined limit/range-finding test) or yellow light (final test). Preparation of test solutions was performed under dimmed light. Various loading rates of the test substance were added to test medium and magnetically stirred for one day. The resulting aqueous mixtures were left to stabilize for 1-1.5 hours where after the clear and colourless WAFs were taken out by siphoning and used for testing. The loading rates for this test were 1.0, 3.2, 10, 32 and 100 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain NIVA CHL 1
Source: In house laboratory culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Stock culture medium was M1 and light intensity was 60-120 μE/m2/s.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
24 mg CaCO3/L
Test temperature:
The temperature of the test medium was 24°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C.
pH:
7.8-8.3
Dissolved oxygen:
No information
Salinity:
Not applicable
Nominal and measured concentrations:
The nominal loading rates were 1.0, 3.2, 10, 32 and 100 mg/L.
The measured initial concentrations were 0.69-0.82, 2.7, 10, 21 and 68-69 mg/L. These concentrations remained stable during the exposure period (96-112% of initial).
Details on test conditions:
The test was carried out as a static test over 72 hours. The test was conducted in 100 mL glass vessels with 50 mL test solution. Test medium was m" with an initial cell density of 1e4 cells/mL. Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking. Light intensity was 85-90 uE/m2/s.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. = 41-70 mg/L
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. = 82-100 mg/L
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% C.L. = 10-41 mg/L
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% C.L. = 19-52 mg/L
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% C.L. = 53-95 mg/L
Details on results:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Aurantiol Pure inhibited growth rate and yield of this fresh water algae species significantly in WAFs prepared at loading rates of 10 mg/L and higher. The EL50 for growth rate inhibition (72h-ERL50) was >100 mg/l and the EL 10 was 59 mg/L. The EL50 for yield inhibition (72h-EYL50) was 72 mg/l with a 95% confidence interval ranging from 53 to 95 mg/l; the corresponding EL10 was 28mg/L. The 72h-NOELR for both growth rate and yield inhibition was 3.2 mg/l.
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.7 mg/L. The EC50 for yield inhibition (72h-EYC50) was 0.56 mg/L.
Reported statistics and error estimates:
An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculation of ELx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rates of the test substance.

Table 1: Percentage inhibition of growth rate (total test period) during the final test

Loading rate (mg/L

Mean

Std. Dev.

n

% inhibition

Control

1.690

0.0229

6

0.0

1.0

1.678

0.0527

3

0.7

3.2

1.678

0.0430

3

0.7

10

1.626

0.0073

3

3.8

32

1.647

0.0375

3

2.5

100

1.314

0.0332

3

22.2

Table 2: Percentage inhibition of yield (total test period) during the final test

Loading rate (mg/L

Mean

Std. Dev.

n

% inhibition

Control

158.425

10.9982

6

0.0

1.0

153.810

24.9716

3

2.9

3.2

153.402

19.8545

3

3.2

10

130.274

2.8713

3

17.8

32

139.536

16.2555

3

11.9

100

50.755

5.1270

3

68.0

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Aurantiol Pure inhibited growth rate and yield of this fresh water algae species significantly in WAFs prepared at loading rates of 10 mg/l and higher. Per definition of the WAF, all effects parameters were related to loading levels. The EL50 for growth rate inhibition (72h-ERL50) was >100 mg/l and the EL10 was 59 mg/L. The EL50 for yield inhibition (72h-EYL50) was 72 mg/l with a 95% confidence interval ranging from 53 to 95 mg/l; the corresponding EL10 was 28mg/L. The 72h-NOELR for both growth rate and yield inhibition was 3.2 mg/l.
The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EL50 and EL10 based on growth rate are used for classification purposes, which were determined in this study to be >100 mg/L and 59 mg/L respectively.
Executive summary:

A study investigating the 72 -hour inhibition effects of Aurantiol Pure on Pseudokirchneriella subcapitata was conducted according to OECD 201. A final test was performed based on the results of a preceding combined limit/range-finding test. Various loading rates of the test substance were added to test medium and magnetically stirred for one day. Water Accommodated Fractions (WAFs) were taken out by siphoning and used for testing. Loading rates were 1.0, 3.2, 10, 32 and 100 mg/l.

Under the conditions of the present study with Pseudokirchneriella subcapitata, Aurantiol Pure inhibited growth rate and yield of this fresh water algae species significantly in WAFs prepared at loading rates of 10 mg/L and higher. The EL50 for growth rate inhibition (72h-ERL50) was >100 mg/L. The EL50 for yield inhibition (72h-EYL50) was 72 mg/L with a 95% confidence interval ranging from 53 to 95 mg/L. The 72h-NOELR for both growth rate and yield inhibition was 3.2 mg/L.

Description of key information

Hydroxycitronellal is being used as an assessment entity for the purposes of the environmental risk assessment of the registered UVCB substance. The rationale for using this substance is provided in the "Assessment Entities" endpoint. The estimated green algae 96h EC50 of 12 mg/L was identified as the most appropriate EC50 for derivation of PNECaquatic for this assessment entity.

Key value for chemical safety assessment

Additional information

For the purpose of classification and labelling, data on the whole substance is considered. A study investigating the 72-hour inhibition effects of Aurantiol Pure on Pseudokirchneriella subcapitata was conducted according to OECD 201. A final test was performed based on the results of a preceding combined limit/range-finding test. Various loading rates of the test substance were added to test medium and magnetically stirred for one day. Water Accommodated Fractions (WAFs) were taken out by siphoning and used for testing. Loading rates were 1.0, 3.2, 10, 32 and 100 mg/l. The concentration of the test substance in the aquatic samples was calculated using the TOC content of the pure test substance, which had been experimetally determined to be 68.20%. A correction was made for the measured amount of TOC in the control. The initial concentrations in samples and reserve samples taken from the WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/l were calculated to be 0.69-0.82, 2.7, 10, 21 and 68-69 mg/l. These concentrations remained stable during the exposure period (96-112% of initial).

Under the conditions of the study with Pseudokirchneriella subcapitata, Aurantiol Pure inhibited growth rate and yield of this fresh water algae species significantly in WAFs prepared at loading rates of 10 mg/l and higher. Per definition of the WAF, all effects parameters were related to loading levels. The EL50 for growth rate inhibition (72h-ERL50) was >100 mg/l and the EL 10 was 59 mg/L. The EL50 for yield inhibition (72h-EYL50) was 72 mg/l with a 95% confidence interval ranging from 53 to 95 mg/l; the corresponding EL10 was 28mg/L. The 72h-NOELR for both growth rate and yield inhibition was 3.2 mg/l.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EL50 and EL10 based on growth rate are used for classification purposes, which were determined in this study to be >100 mg/L and 59 mg/L respectively.