Essential oil Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction (indicative)

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:

experimental study

Adequacy of study:

weight of evidence

Study period:

07-20 November 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This GLP study was performed according to OECD Guideline 201 with the following deficiencies: old WAF method - dispersions were stirred for 3 hours and then filtered.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The Swiss GLP Monitoring Authorities (inspected on 05-09 & 26-30 November 2007 / signed on 12 November 2008)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Pale yellow liquid

Analytical monitoring:

yes

Details on sampling:

Analysis of the test Item concentrations:
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 analyzed immediately after sampling. Immediately after sampling, acetone was added to each sample (1% of acetone per sample volume).
The concentrations of the test item were determined in the duplicate test medium samples from all test concentrations. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared (table 6.1.5/1).
The dispersions were stirred for 3 hours at room temperature in the dark to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrates were tested as WAFs. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of volatile components of the test item during filtration.
The stirring period of 3 hours was chosen based on the results of a pre-experiment (non-GLP) in which the maximum concentration of the different compounds in the test media was reached after the stirring period of 3 hours.
The test media were prepared just before the start of the test.
- Controls: Test water without test item

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen/Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

Water temperature during the test was 22 °C

pH:

At the start of the test, the pH measured in the treatments was between 8.0 and 8.1. At the end of the test, pH values of 8.8 to 8.9 were measured (Table 6). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth despite the test media were stirred during the test.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Water accommodated fractions (WAFs) with the loading rates 0.10, 0.33, 1.0, 3.3 and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50-mL Erlenmeyer flasks
- Type: closed; test flasks were made tight with glass stoppers to avoid losses of the volatile substance.
- Material, size, headspace, fill volume: Since the test item was determined to be volatile (according to the results of a pre-experiment, non-GLP), the test was performed in 50-mL Erlenmeyer flasks completely filled with about 50 mL test medium.
- Aeration: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: At the start of the test, 10000 cells/mL were inoculated; the algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae were cultivated and tested in synthetic test water, prepared according to the test guidelines, but modified according to the International Standard ISO 14442 as the test was performed in a closed test system. The concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer were added to keep the pH in the test media as constant as possible.

OTHER TEST CONDITIONS
- The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The mean measured light intensity at the level of the test solutions was approximately 7300 Lux (range: 6600 to 7800 Lux, measured at nine places in the experimental area).

EFFECT PARAMETERS MEASURED:
Determination of Algal Biomass:
- A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced.
- The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate.
- At the end of the test, a sample was taken from the control and from the loading rate of nominal 3.3 mg/L. The shape and size of the algal cells were visually inspected. This loading rate was chosen since, at the highest nominal loading rate of 10 mg/L, the algal cell density was too low for a reliable examination.
- Water Quality Criteria: The pH was measured and recorded in each treatment at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- The selection of the loading rates was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water (non-GLP). The enlarged spacing factor of 3.2 between the test concentrations was chosen, because according to the results of the range-finding test the concentration-effect relationship was rather flat and, thus, a large concentration range had to be tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 3.8 – 4.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 8.9 – 9.4 mg/L

Duration:

72 h

Dose descriptor:

other: The highest tested loading rate without observed effect

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- The test item had a significant inhibitory effect on the average growth rate of the algae after the test period of 72 hours at the concentration of 10 mg/L and on yield at the concentration of 3.3 mg/L and at all higher test concentrations (results of Williams-tests, one-sided, α = 0.05). Thus, the concentration of 3.3 mg/L was determined to be the 72-hour LOEC.
- The 72-hour NOEC was determined to be 1.0 mg/L, since up to and including this test concentration the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.
- 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 3.3 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.
- No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the historical range of Harlan Laboratories (72-hour EC50 for the growth rate: 1.20 mg/L (RCC Study No. B83755), range of the 72-hour EC50 for the growth rate from 2000 to 2008: 0.71–1.74 mg/L).

Reported statistics and error estimates:

The 72-hour ELC10, EL20 and EL50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams tests.

Analysis of concentrations: At the start of the test, the measured test item concentrations (based on the sum of four peaks) in the test media with the loading rates of 0.10, 0.33, 1.0, 3.3 and 10 mg/L were between 0.006 and 0.69 mg/L. At the end of the test, the measured concentrations in the test media were between < LOQ and 0.31 mg/L. Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

 

Table 6.1.5/2: Average Growth Rates (μ)

Loading rate (mg/L)	Average growth rate μ (day^-1) and inhibition of μ (Ir)
	0-24 h		0-48 h		0-72 h
	μ	Ir(%)	μ	Ir(%)	μ	Ir(%)
Control	1.098	0.0	1.695	0.0	1.737	0.0
0.10	1.255	-14.3	1.697	-0.1	1.727	0.6
0.33	1.262	-15.0	1.714	-1.1	1.724	0.8
1.0	1.181	-7.5	1.706	-0.6	1.733	0.2
3.3	1.088	0.9	1.644*	3.0	1.655	4.7
10	0.642*	41.5	0.751*	55.7	0.762*	56.1

*: mean value significantly lower than in the control (according to Williams-tests, one-sided, α = 0.05)

 

Table 6.1.5/3: Yield (Y)

 

Loading rate (mg/L)	Yield Y (x 10^3) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	1.893	0.0	28.491	0.0	168.431	0.0
0.10	2.316	-22.3	28.623	-0.5	164.261	2.5
0.33	2.365	-24.9	29.624	-4.0	168.594	-0.1
1.0	2.094	-10.6	29.138	-2.3	166.160	1.3
3.3	1.830	3.4	25.527*	10.4	133.721*	20.6
10	0.859*	54.6	3.357*	88.2	8.360*	95.0

*: mean value significantly lower than in the control (according to Williams-tests, one-sided, α = 0.05)

 

Validity criteria:

In the control the biomass increased by a factor of 184 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.

The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 34%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.5%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions and based on the loading rates, the ErL10 and ErL50 values were calculated to be 4.2 mg/L (95% CI: 3.8-4.5 mg/L) and 9.1 mg/L (95% CI: 8.9-9.4 mg/L), based on growth rate, respectively.

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 72-hour static test according to OECD Guideline 201/ EU method C.3 and in compliance with GLP.

 

In order to test the toxicity of the multi-component test item to the algae, water accommodated fractions (WAFs) with different loading rates of the test item were prepared and tested. WAFs with the following loading rates of the test item were tested: 0.10, 0.33, 1.0, 3.3 and 10 mg/L. A control was run in parallel.

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 3 hours at room temperature in the dark to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters and the undiluted filtrates were tested as WAFs. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of volatile components of the test item during filtration. The stirring period of 3 hours was chosen based on the results of a pre-experiment (non-GLP) in which the maximum concentration of the different compounds in the test media was reached after the stirring period of 3 hours.

 

At the start of the test, the measured test item concentrations (based on the sum of four peaks) in the test media with the loading rates of 0.10, 0.33, 1.0, 3.3 and 10 mg/L were between 0.006 and 0.69 mg/L. At the end of the test, the measured concentrations in the test media were between < LOQ and 0.31 mg/L. Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

 

The test item had a significant inhibitory effect on the average growth rate of the algae after the test period of 72 hours at the concentration of 10 mg/L. 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 3.3 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. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

 

Under the test conditions and based on the loading rates, the ErL10 and ErL50 values were calculated to be 4.2 mg/L (95% CI: 3.8-4.5 mg/L) and 9.1 mg/L (95% CI: 8.9-9.4 mg/L), based on growth rate, respectively.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

March 26th, 2019

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

Justification for type of information:

1. SOFTWARE
iSafeRat® HA-QSAR toolbox

2. Model version
iSafeRat® holistic HA-QSAR v1.8

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

4. APPLICABILITY DOMAIN
See attached QPRF

5. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not applicable

Remarks:

(QSAR model)

Principles of method if other than guideline:

A calculation method was used to predict the inhibition of algal growth exposed to the test item Essential oil of Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction (South Africa), a Natural Complex Substance, using the WAF method. This calculation method predicts
the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European
Commission, 2008) adapted for testing of a mixture using the WAF method. The criterion measured was the median effective loading rate of the mixture ErL50 (Median Effective Loading for specific growth rate), a statistically derived loading rate which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.T
The growth inhibition of algae was determined using a validated calculation method for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis (Bauer et al., 2018)). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas,
2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). The QSAR model is based on validated data from a training set of 40 chemicals derived from 72-hour test on algae, for which the concentrations of the test item had
been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the nonbioavailable fraction, this approach is based on validated data derived from 72-hour ErL50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The calculation method used in this study is based on toxic additivity principle. That means the toxic parts of each constituent are added up. Therefore the constituents considered within the mixture should act with a similar MechoA. The MechoA of the consituents are determined using the methodology described by Bauer et al., (2018).
Since the constituents of the test item act with the same MechoA (1.1), the calculation method is directly applicable. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European Commission, 2008). This approach uses a thermodynamically based module to calculate the water concentrations and the individual toxicity of each constituent is first derived from a QSAR model validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). Then the effective loading rate of the mixture to the test organisms (i.e. Pseudokirchneriella subcapitata) is determined as a Water Accommodated Fraction (WAF) test (OECD, 2002). Method detailed within the position paper written by Bicherel and Thomas (2014).

Step 1. Determination of the water solubility of each individual constituent:
The iSafeRat® mixture module first uses phase equilibrium thermodynamics to determine the water solubility of the individual constituents when they are together within the mixture and considering the non-ideality of the solution (i.e. by taking into account potential intermolecular interactions).

Step 2. Determination of the Analytically Measurable Aqueous Phase (AMAP).
Using the model, the specific concentration of each constituent in the WAF can be accurately determined at any loading rate taking into account the original composition of the mixture and the new corrected limit of solubility of each constituent. The sum of these concentrations, the AMAP, is equivalent to the concentration of all constituents that would be measured in a WAF test. For a given loading rate, the loaded concentration of each constituent is compared to its respective solubility limit. If one is higher than the solubility limit then it is considered that the concentration of the constituent will be equal to its solubility limit. If lower, the true loading concentration is considered.Provided this adapted water solubility limits for each consitutents. analytically measurable concentrations in aqueous solutions expected from known WAF loading rates of characterised mixtures can be calculated.

Step 3. Determination of the bioavailable toxic fraction of each constituent.
Each concentration of constituent is then decreased by a nonbioavailability factor internally determined by KREATiS.This factor, based on the hydrophobicity of each constituent, takes into account the tendency of a compound to rather partition with itself than with water.

Step 4. Calculation of the global activity of the WAF and determination of the toxic loading rate.
After removing the non-bioavailable fraction, the concentration of each constituent is converted into activities, then they are summed assuming the principle of additivity or Concentration Addition (CA) (Loewe & Muischnek, 1926). This principle implies that each constituent exerts an analogous mode of action and can be replaced by another constituent as long as the global toxicity is still the same (Backhaus et al., 2003).


More details are availble in QPRF.

GLP compliance:

no

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

other: algae spp.

Details on test organisms:

Not Applicable

Test type:

other: QSAR method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

Not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperature varied from approximately 20 to 25 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the ErC50 values found in experimental data.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

Not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF method

Details on results:

Not applicable

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not determinable

No additionnal information

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the growth inhibition of algae has been determined using the calculation method for mixtures based on WAF studies. The test item constituents which have been considered fall individually within the applicability domain of the model used to determine the toxicity value on algae (as the 72h-ErL50). Therefore, the final result for the test item can be considered valid for use in risk assessment and classification and labelling.
The 72h-ErL50 of the test item to algae was predicted as 6.5 mg/L.

Executive summary:

A calculation method was used to predict the inhibition of algal growth exposed to the test item Essential oil of Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction (South Africa), a Natural Complex Substance, using the WAF method. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European Commission, 2008) adapted for testing of a mixture using the WAF method. The criterion measured was the median effective loading rate of the mixture ErL50 (Median Effective Loading for specific growth rate), a statistically derived loading rate which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

The growth inhibition of algae was determined using a validated calculation method for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis (Bauer et al., 2018)). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). The QSAR model is based on validated data from a training set of 40 chemicals derived from 72-hour test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the nonbioavailable fraction, this approach is based on validated data derived from 72-hour ErL50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity value anticipated during a 72-hour ErL50 study on algae based on the WAF method: is 6.5 mg/L.

Description of key information

QSAR model, iSafeRat holistic approach v1.8, Weight of Evidence, validity 2:

WAF, 72h-ErL50 = 6.5 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

6.5 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, a weight of evidence approach has been considered with one experimental study and one QSAR prediction.

The experimental study was performed one the standard quality (Item 2) according to OECD Guideline 201 with GLP statement. The influence of the registered substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test. Water Accommodated Fractions (WAFs) with different loading rates (0.10, 0.33, 1.0, 3.3 and 10 mg/L) of the test substance were prepared and tested. A control was run in parallel. At the start of the test, the measured test substance concentrations (based on the sum of four peaks) in the test media with the loading rates of 0.10, 0.33, 1.0, 3.3 and 10 mg/L were between 0.006 and 0.69 mg/L. At the end of the test, the measured concentrations in the test media were between < LOQ and 0.31 mg/L. Since WAF of the test substance were tested, all reported biological results were based on the loading rates of the test substance.

The test substance had a significant inhibitory effect on the average growth rate of the algae after the test period of 72 hours at the concentration of 10 mg/L. 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 3.3 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. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. 

Under the test conditions and based on the loading rates, the ErL10 and ErL50 values were calculated to be 4.2 mg/L (95% CI: 3.8-4.5 mg/L) and 9.1 mg/L (95% CI: 8.9-9.4 mg/L), based on growth rate, respectively.

The QSAR prediction was performed on the quality with a high level of alpha pinene (Item 5): a calculation method was used to predict the inhibition of algal growth using the WAF method. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European Commission, 2008) adapted for testing of a mixture using the WAF method. The criterion measured was the median effective loading rate of the mixture ErL50 (Median Effective Loading for specific growth rate), a statistically derived loading rate which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

The growth inhibition of algae was determined using a validated calculation method for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis (Bauer et al., 2018)). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). The QSAR model is based on validated data from a training set of 40 chemicals derived from 72-hour test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the nonbioavailable fraction, this approach is based on validated data derived from 72-hour ErL50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. The toxicity value anticipated during a 72-hour ErL50 study on algae based on the WAF method is 6.5 mg/L for the new quality (high level of alpha pinene).

In conclusion, toxicity values during a 72 -hour ErL50 study on algae based on the WAF method are 9.1 and 6.5 mg/L for both qualities. Despite the difference of level of alpaha pinene between both qualities, toxicity values are in the same range for algae. Key value of the quality with higher level of alpha pinene is considered as worst case with an 72-hour ErL50 of 6.5 mg/L.