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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 to 08 February 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 201. However, pH level in the control and in the limit test varied more than 1.5 units at the end of the test and chemical analyses revealed severe test item losses in the presence and absence of algae. This study is considered reliable with restrictions.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH level in the control and in the limit test varied more than 1.5 units at the end of the test
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
pH level in the control and in the limit test varied more than 1.5 units at the end of the test
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
2013-01-11
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for analysis were taken from the control and the limit test concentration (from a replicate of each treatment with and without algae dedicated exclusively to chemical analyses).
- Sampling method: Samples were taken At the start (t = 0 h) and every day thereafter until the end of the test. Approximately 500 µL were taken for each sample and analysed by HPLC-DAD.
- Sample storage conditions before analysis: Samples were analysed directly after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Given the volatility of the test item (vapour pressure: 530 Pa at 25 °C), precautions were taken to minimize the volatilisation and to maximise the solubilisation during the preparation of test solutions in accordance with recommendations given in the OECD document on testing difficult substance (OECD, 2000) and the OECD 201 Guideline
- Method: the stock and test solutions were prepared under closed conditions and gently stirred to avoid production of a dispersion. A stock solution was prepared by slow-stirring. The mixing vessel (1 L) was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. A magnetic stirring bar was placed in the vessel and 1 L of the test medium was added. This is to use a maximum volume and to minimize headspace whilst maintaining optimum surface contact between test item and the water. Then an excess of the test item (approximately 4 g) was carefully added directly to the surface of the test water. Mixing was initiated with the vortex in the centre extending maximally around 10 % vessel depth from the top to the bottom of the vessel. The stirring speed was kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 h of gentle stirring and a few minutes to settle (ca. 15 minutes), the saturated aqueous phase was taken out of the drain port. The first 100 mL were discarded then the stock solution was directly added into test flasks with a fixed amount of inoculum to obtain a cell density of 5000 cells/mL/vessel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test sample formed a clear colourless solution and appeared to be completely soluble when mixed with test water.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source: Museum National d'Histoire Naturelle, Paris, bred in the Laboratory under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 2-4 days
- Culturing media and conditions: Same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Study initially over 72 h. However, due to dissipation of the tested substance, it was considered more relevant to consider the exposure at 48h, a recommendation according to OECD 201 as long as the validity criteria for the study were still met.
Post exposure observation period:
No
Hardness:
No data
Test temperature:
22.4- 23.5 °C
pH:
8.06-9.90.
It was proposed that the cause of the pH increases was due to the substantial algal growth in conjunction with closed conditions used in the test.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentration: limit test, at the solubility/saturation limit of the test item.
- Measured concentration (at t = 0 h): 0.905 mg/L (biotic system) or 2.259 mg/L (abiotic system). See table 6.1.5/1.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks (100 mL) sealed with a fritted glass stopper
- Type: Closed
- Fill volume: 50 mL of algal suspension per replicate
- Shaking: During incubation, the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 5000 cells/mL
- Control end cells density: 567000 ± 70000 cells/mL
- No. of vessels per concentration (replicates): Six
- No. of vessels per control (replicates): Six
- Moreover, a replicate of each treatment with and without algae was prepared in order to assess potential bioaccumulation and/or adsorption effects of the test item by P. subcapitata during the test period and to determine maintenance of actual concentrations by chemical analyses.

GROWTH MEDIUM
- Standard medium used: Yes; original medium of OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Original medium of OECD TG 201, prepared using sterilised water
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured continuously in the growth chamber, over the study period and pH was recorded at the beginning and at the end of the test in one vessel per concentration and the control (same vessel at t = 0 and 72 h).

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 5920-6935 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Previous GLP non-limit test
- Test concentrations: 0.05, 0.10, 0.22, 0.46, 0.96 and 2.0 mg/L (nominal)
- Results used to determine the conditions for the definitive study: In the non-limit test, except the first day at the higher test concentrations, no significant inhibition of the growth rate was recorded the remainder of the test for all test concentrations. The chemical analyses revealed that test item concentrations were not stable during the test period. In view of these results, it was decided to perform a limit test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (non-concurrent)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.131 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
recalculated value
Details on results:
- A slight inhibition (38.5 %) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8 % at t = 48 h and 0 % at t = 72 h).
- Unusual cell shape: Microscopic examination of the shape of the algal cells every day did not show any difference between algae that had been growing at the limit test concentration and the algal cells in the control.
- Refer tables 6.1.5/2, 6.1.5/3 and 6.1.5/4 for more details

Regarding the test analyses results and taking into account the possibility that the organisms may not have been exposed to the test substance throughout the study, a more conservative exposure regime has been considered as a worst case scenario. A more conservative NOEC value for specific growth rate of 0.131 mg/L has been determined. This endpoint is supported by a High Accuracy QSAR (HA-QSAR) prediction using iSafeRat® with a worst-case ErC50 value of 0.31 mg/L
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Historical data (July 2012): ErC50 (72 h) = 0.96 mg/L
Reported statistics and error estimates:
- Statistical analysis was performed using software ToxRat® Professional.

ANALYTICAL RESULTS:

- Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day [biotic system: 0.905 mg/L at t = 0 h and between LOD (0.03 mg/L) and ( LOQ (0.1 mg/L) at t = 24h; abiotic system: 2.259 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24 h].

- These test item losses were considered to be associated to some degree with metabolism and/or adsorption effects of the test item by P. subcapitata, but given the significant test item losses also observed in the abiotic system, it would be more likely due to a photodegradation phenomenon or another abiotic degradation characteristic of the test item.

However, ecotoxicological results were recalculated in order to take into account losses of test item.

- Refer table 6.1.5/1 for more details

Table 6.1.5/1:Concentrations analysed (mg/L) of the test substance in test water - limit test: biotic and abiotic system

Treatment

Start (t = 0 h)

t = 24 h

t = 48 h

End (t = 72 h)

Biotic system*

Control

< LoQ

< LoQ

< LoQ

< LoQ

Limit concentration

0.905

LoD < C < LoQ#

LoD < C < LoQ#

LoD < C < LoQ#

Abiotic system

Limit concentration

2.259

LoD < C < LoQ#

0.108

0.104

* Preparation of samples for quantification of biotic system requested a transfer step in all-glass hemolysis tubes for centrifugation in contrast to abiotic system, which could explained the difference in concentration at t= 0 h found between biotic and abiotic system.

# At this time, the limit test concentration was below the LOQ (0.1 mg/L) but above the LOD (0.03 mg/L)

As substance concentration was between LoD and LoQ at T24h and T48h, there is proof that the algae must have been exposed to the test substance throughout the study (used for determination of the endpoint).

BIOLOGICAL RESULTS:

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10^4)

 

Replicate

Control

Limit concentration

T = 24h

1

2

3

4

5

6

4.0

3.6

4.0

2.8

2.0

4.0

1.2

2.0

2.4

2.0

1.2

1.2

Mean

Std. Dev.

3.4

0.83

1.7

0.53

T = 48h

1

2

3

4

5

6

22.8

24.4

16.4

22.4

24.8

20.0

16.8

17.6

18.0

14.0

11.2

18.4

Mean

Std. Dev.

21.8

3.15

16.0

2.83

T = 72h

1

2

3

4

5

6

52.8

52.4

55.2

49.2

63.2

67.2

56.0

54.0

58.0

59.2

54.0

56.4

Mean

Std. Dev.

56.7

7.00

56.3

2.10

Table 6.1.5/3: Yield (Y) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

Y

% I

Y

% I

Y

% I

Control

2.900

0

21.300

0

56.167

0

Test item

1.167

59.8

15.500

27.2

55.767

0.7

Table 6.1.5/4: Growth rate (G) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

G

% I

G

% I

G

% I

Control

1.887

0

1.883

0

1.575

0

Test item

1.161

38.5

1.725

8.4

1.574

0

Recalculation of endpoint-value:

The concentration measured in the abiotic sample are close to the limit of solubility of the tested substance (2.75 mg/L).

The lower concentration measured in the biotic sample may be explained by:

      - Loss due to the volatility and the additional manipulation of the samples prior to analysis due to the necessity to remove the algae by centrifugation. Test solution was transferred in centrifugation tubes to remove algae before analysis. The substance has a high Henry Constant and a few minutes would be enough to cause significant loss of material under these conditions.

      - Adsorption of the substance on algae but this does not imply substance loss from the test system. As abiotic samples do not undergo this step, we can consider that loss of substance occurred mostly in the biotic samples and that losses in the abiotic samples were much lower. Therefore the most appropriate concentration values closest to the actual exposure concentrations would be the abiotic sample concentrations.

The concentration of 2.259 mg/L is a better representation of the real exposure of organisms and the concentration of 0.905 mg/L represents the minimum exposure concentration (after significant volatility loss which occurred outside of the study during sample analysis).

The calculation of toxicity data based on the concentration of 0.905 mg/L is therefore considered as a worst-case value.

 To express a more conservative exposure of algae, the exposure concentration was calculated with the biotic samples. According to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, ENV/JM/MONO (2000)6, “ When the substance is detected but not quantified, it may be a good practice to use half of the limit of quantification.”At 24 and 48 h the substance is detected, the concentration is assumed to be 0.05 mg/L (half of the quantification limit, close to the detection limit). The exposure concentration over 48h was 0.131 mg/L, calculated as the mean geometric of 0.905, 0.05 and 0.05 mg/L (concentrations at 0, 24 and 48h).

  As inhibition is not significant at 48 h we can consider a NOEC of 0.131 mg/L.

Validity criteria fulfilled:
yes
Remarks:
control cultures (0-48 h): cell concentration increased by 44 folds; mean CV for section by section specific growth rate was 19 %; CV for average specific growth rate was 4.1 %
Conclusions:
The 48-h NOEC value of (-)-alpha pinene on Pseudokirchneriella subcapitata were estimated to be 0.131 mg/L after recalculation taking into account losses of test item during the experimental phase of the study.
This GLP study was performed according to OECD Guideline No. 201 and validity criteria were fulfilled.
Executive summary:

An algal toxicity study was performed on Pseudokirchneriella subcapitata with (-)-alpha-pinene in accordance with GLP and OECD Guideline OECD 201.

A preliminary test was conducted: algae were exposed to (-)-alpha pinene at nominal concentrations of 0.05, 0.10, 0.22, 0.46, 0.96 and 2.0 mg/L for 72 h. Except the first day at the higher test concentrations, no significant inhibition of the growth rate was recorded during the remainder of the test for all test concentrations.

In view of these results, a definitive test was conducted: algae were exposed to the test item at its solubility/saturation limit concentration (six replicate flasks per test concentration and control) for 72 h, in closed conditions under constant illumination and shaking at a temperature of 22.4 -23.5 °C. Measurement of biomass and growth inhibition was done by determination of cell densities at 24, 48 and 72 h using counting chamber. Samples taken from each treatment (biotic and abiotic medium) were analysed at the start and every day thereafter until the end of the test in order to determine maintenance of actual concentrations.

All the possible precautions described in the OECD document on testing difficult substance (OECD, 2000) and the OECD 201 Guideline were taken into account to conduct the study.

A slight inhibition (38.5%) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8% at t = 48 h and 0% at t = 72 h).

Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day [biotic system: 0.905 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24h; abiotic system: 2.259 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24 h]. The concentration measured in the abiotic sample are closed to the limit of solubility of the tested substance (2.75 mg/L).

To express a more conservative exposure of algae, the exposure concentration was calculated with the biotic samples. The concentration of 2.259 mg/L is a better representation of the real exposure of organisms and the concentration of 0.905 mg/L represents the minimum exposure concentration (after significant volatility loss which occurred outside of the study during sample analysis).

At 24 and 48 h the substance is detected, the concentration is assumed to be 0.05 mg/L (half of the quantification limit, close to the detection limit).

The exposure concentration over 48 h was 0.131 mg/L, calculated as the geometric mean of 0.905, 0.05 and 0.05 mg/L (concentrations at 0, 24 and 48h).

Therefore, the 48-h NOEC values of (-)-alpha pinene on Pseudokirchneriella subcapitata was estimated to be 0.131 mg/L.  

Validity criteria:

The cell density in the control cultures should have increased at least by a factor of 16 within exposure period (in this study a 44-fold increase was observed within 48 h).

The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35% (in this study 19% for 0-24h and 24-48h).

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (in this study 4.1% within 48h).

Therefore the validity criteria have been fulfilled and the study is considered valid over 48 h.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2017-03-03 to 2017-03-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR value. The substance falls into applicability domains of the model QSAR
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
QSAR model
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
QSAR model
Principles of method if other than guideline:
The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 72-hour ErC50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.
GLP compliance:
no
Specific details on test material used for the study:
No data.
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
Not applicable.
Test organisms (species):
not specified
Details on test organisms:
No data
Test type:
other: QSAR model
Water media type:
not specified
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Not applicable
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Not applicable
pH:
Not applicable
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Not applicable
Details on test conditions:
Not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.31 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 0.27 – 0.36 mg/L
Details on results:
Not applicable
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
95% CL [0.27-0.36] QSAR statistical parameters are given in the QMRF and the QPRF.

None.

Validity criteria fulfilled:
yes
Conclusions:
72 h-ErC50 for (-)-α-pinene = 0.31 mg test item/L with 95 %-Confidence Limit of 0.27-0.36 mg test item/L.

Based on the results of this study, (-)-α-pinene is classified:
- dangerous for the environment according to Directive 67/548/EEC " R50 Very toxic to aquatic organisms, and R53 May cause long-term adverse effects in the aquatic environment"
- and hazardous to the aquatic environment under Regulation (EC) No 1272/2008 (CLP) - Acute Category 1 and Chronic Category 1.
Executive summary:

A QSAR model was used to predict the inhibition of growth to algae of test item (-)-α-pinene. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008. The criterion was the ErC50 (Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours. 

The QSAR determination of the algae growth inhibition was based on validated data derived from 72 h ErC50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test periods.

 

The 72 h-ErC50 (based on growth) is predicted as 0.31 mg/L with 95%-Confidence Limit between 0.27 and 0.36 mg test item/L.

 

This toxicity study is considered acceptable and satisfies the guideline requirements for growth inhibition of algae study.

 

Results Synopsis

Test Type: QSAR model

ErC50: 0.31 mg test item/L 95 %-C.I.: 0.27-0.36 mg test item/L

Description of key information

The recalculated 48h-NOErC for freshwater algae for (-)-alpha-pinene is 0.131 mg/L.


Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.131 mg/L

Additional information

An algal toxicity study was performed onPseudokirchneriella subcapitatawith (-)-alpha-pinene in accordance with GLP and OECD Guideline OECD 201.

A preliminary test was conducted: algae were exposed to (-)-alpha pinene at nominal concentrations of 0.05, 0.10, 0.22, 0.46, 0.96 and 2.0 mg/L for 72 h. Except the first day at the higher test concentrations, no significant inhibition of the growth rate was recorded during the remainder of the test for all test concentrations.

In view of these results, a definitive test was conducted: algae were exposed to the test item at its solubility/saturation limit concentration (six replicate flasks per test concentration and control) for 72 h, in closed conditions under constant illumination and shaking at a temperature of 22.4 -23.5 °C. Measurement of biomass and growth inhibition was done by determination of cell densities at 24, 48 and 72 h using counting chamber. Samples taken from each treatment (biotic and abiotic medium) were analysed at the start and every day thereafter until the end of the test in order to determine maintenance of actual concentrations.

All the possible precautions described in the OECD document on testing difficult substance (OECD, 2000) and the OECD 201 Guideline were taken into account to conduct the study.

A slight inhibition (38.5%) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8% at t = 48 h and 0% at t = 72 h).

Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day [biotic system: 0.905 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24h; abiotic system: 2.259 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24 h].The concentration measured in the abiotic sample are closed to the limit of solubility of the tested substance (2.75 mg/L).

To express a more conservative exposure of algae, the exposure concentration was calculated with the biotic samples.The concentration of 2.259 mg/L is a better representation of the real exposure of organisms and the concentration of 0.905 mg/L represents the minimum exposure concentration (after significant volatility loss which occurred outside of the study during sample analysis).

At 24 and 48 h the substance is detected, the concentration is assumed to be 0.05 mg/L (half of the quantification limit, close to the detection limit).

The exposure concentration over 48 h was 0.131 mg/L, calculated as the geometric mean of 0.905, 0.05 and 0.05 mg/L (concentrations at 0, 24 and 48h).

Therefore, the 48-h NOEC values of (-)-alpha pinene onPseudokirchneriella subcapitatawas estimated to be 0.131 mg/L.  

In order to support the experimental result, iSafeRat® High-Accuracy QSAR models were used on (-)-alpha-pinene. The model for non-polar narcosis (or MOA1 according to Verhaaret al.;1992) dedicated to algae toxicity was constructed using high quality data for water solubility and algal growth inhibition test for 40 substances with the same mode of action. This mechanism of toxicity is based on the principle of biological membrane disruption by a reversible accumulation of chemicals into the membrane induced by hydrophobic interactions. (-)-αPinene as an alkene (terpene) is attributed to the class of non-polar narcotic compounds (MOA1).

All chemical groups within the molecular structure are taken into account by the model.

The water solubility of (-)-α pinene was predicted at 3.11 mg/L (or -4.64 in log (mol/L) using the iSafeRat® solubility module. This value was given as the input in the QSAR model to determine the algae toxicity. The water solubility value given as the input to the iSafeRat® Holistic HA-QSAR model falls just beyond the regression domain of the model between a log water solubility (in log (mol/L)) of -4.38 to 0.49. It falls in an intermediate domain between a log water solubility (in log (mol/L)) of - 9.34 to -4.38 where baseline toxicity cannot be experimentally measured accurately because the thermodynamic equilibrium is unlikely to be reached within the exposure duration of the study. Therefore the toxicity is likely to be greater than the water solubility limit. Based on a statistical k-NN (k Nearest Neighbours) approach (k = 3), the predicted toxicity of the test item can still be extrapolated using the linear regression since the closest neighbours have a ErC50 below their solubility limit. It should be noted that the regression can therefore be considered a worst case toxicity scenario for the algae under these conditions.

The 72h-ErC50 of (-)-α pinene to algae was extrapolated as equal to 0.31 mg/L with a 95% confidence interval (α = 0.05) of 0.27 to 0.36 mg/L.

It is considered that the experimental OECD 201 study fulfils the information requirement laid down in Annex VII, Section 9.1.2 of the REACH regulation and therefore it is considered as key study. The 48 h-NOErC for the registered substance to freshwater algae is 0.131 mg/L.

Therefore, the experimental NOEC value is valid for key value for chemical safety assessment.