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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:
05 Dec 2017 - 22 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test item information
Identification: Cedarwood Texas oil crude
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: LS160616
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 15 June 2018 (expiry date)

Additional information
Test item: 207802/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Cedarwood Texas Oil
CAS Number: 91722-61-1
Analytical monitoring:
yes
Remarks:
TOC measurement
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h and t=72 h
Volume 40 mL
Storage Samples were stored in a refrigerator (2-8°C) until analysis.
At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.
Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of Cedarwood Texas oil crude tested was a yellow liquid. The test item was a UVCB substance and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with loading rates individually prepared ranging between 0.46 and 100 mg/L. Exact amounts of test item were added to test medium without HEPES buffer. A 2-day period of magnetic stirring in closed vessels with minimal headspace and in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool* and used as test concentrations. Microscopic observation of the WAFs showed that they did not contain undissolved test material. The final test solutions were clear and colourless, except for the highest WAF in the full test, which was observed to be hazy. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.24 mL) was spiked to each vessel.

Solutions for analysis of TOC concentrations:
 At the start of the test: samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
 At the end of the exposure period: volumes of 40 mL of each WAFs were added to vessels with no algae at the start of the test and used as solutions for analysis of TOC concentrations at the end of the test. These vessels were not spiked with HEPES buffer. This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.
Any residual volumes were discarded.

*Glass wool was not used in the range-finding test as no haziness of solutions was observed.
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.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

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.
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator varied between 22 and 23°C.
pH:
t=0h : 7.3 - 7.4
t =72h: 7.3 - 7.8
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).
Nominal and measured concentrations:
Nominal: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.

TOC measurements:
Loading rates: Control, 1.0, 3.2, 10, 32 and 100 mg/L.
TOC t=0h : n.q, n.q, n.q, 1.9, 4.0 and 16 mg/L
TOC t=72h: n.q, n.q, n.q, n.q, 2.5 and 7.3mg/L
n.q. – not quantifiable

The TOC content of the test item was determined to be 83.52%
Details on test conditions:
TEST SYSTEM
- Cedarwood Texas oil crude: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
- Controls: Test medium without test item or other additives.
- Replicates:
- 3 replicates of each test concentration
- 6 replicates of the control
- 2 extra replicates of each test concentration and the control without algae and HEPES for sampling purposes
- 1 extra replicate of each test item concentration
- Test duration: 72 hours
- Test type: Static
- Test vessels: 40 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
- Medium: Adjusted M2
- Cell density: An initial cell density of 1 x 10^4 cells/mL.

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 75 to 80 µE.m-2.s-1.
- Incubation: Airtight closed vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENT AND RECORDINGS
- pH: At the beginning and at the end of the test.
- Temperature of medium: Continuously in a temperature control vessel.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the control and the highest concentration

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Because the haziness of the highest WAF could disturb spectrophotometric measurement, algal density was determined by use of a microscope and a counting chamber throughout the test.

RANGE- FINDING TEST:
A range-finding test was performed to provide information about the range of loading rates to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Exponentially growing algal cultures were exposed to WAFs prepared at loading rates of 0.46, 1.0, 10 and 100 mg/L and to a control.
- Three replicates were tested per loading rate and three replicates in the control group.
- pH was only measured in the control and the highest test loading rate.
- Cell densities were determined only at the end of the exposure.
- At the end of the test algae were not observed to verify a normal and healthy appearance.
- Cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates as a background for treated solutions.

- Results used to determine the conditions for the definitive study: The expected EL50 for growth rate inhibition was above a WAF prepared at a loading rate of 100 mg/L. The expected EL50 for yield was between 1.0 and 10 mg/L. Because the obtained dose-response was shallow, it was decided for the full test, to use a range of loading rates to cover the EL10/EL50, rather than to focus on the NOEL.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 35-48
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 11-19 mg/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 4.8-11
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance,
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
Remarks on result:
other: based on biological significance
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95%CL: 4.5 - 5.6
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
0.57 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CL: 0.43-0.72
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Measured Test Item Concentrations:
Samples taken from all groups were analysed. A clear increase in measured TOC concentrations was observed in WAFs of 10, 32 and 100 mg/L, indicating proper preparation of WAFs. At the lower WAFs and in the control, no TOC was detected. The measured concentrations were higher than in the range-finding test, which is also in line with the observation that the highest WAF was hazy in the final test, while clear in the range-finding test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Inhibition of Growth Rate and Inhibition of Yield:
A dose-related increase of inhibition of growth rate and yield was observed at loading rates of 3.2 to 100 mg/L. The effect observed at the highest concentration for the growth rate was higher than in the range-finding test, but in line with the higher measured TOC concentration. A statistically significant inhibition of both, growth rate and yield was observed at all WAFs tested. The inhibition of growth rate observed at the two lowest WAFs was considered biologically not relevant, i.e. <10%. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all WAFs when compared to the control.

Acceptability of the Test:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 208).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 22%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.1%).
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOEL and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure (growth rate) or Trend analysis by Contrasts (Monotonicity of Concentration/Response) (yield); both α=0.05, one-sided, smaller). Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of ELx values was based on Weibull analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal loading rates of the test item. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Growth Rate (µ, day-1) And Percentage Inhibition for the Total Test Period

Cedarwood Texas oil crude
Loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.778

0.0189

6

 

1.0

1.641#

0.0056

3

7.7

3.2

1.647#

0.0200

3

7.3

10

1.423*

0.0143

3

20

32

1.197*

0.0393

3

33

100

0.189*

0.1155

3

89

* - effect was statistically significant,#effect was statistically significant but biologically not relevant (<10%)

Yield (x104Cells/mL) And Percentage Inhibition For The Total Test Period


Cedarwood Texas oil crude

Loading rate (mg/L)

Mean

Std. Dev.

n

% Inhibition

Control

206.5

12.06

6

 

1.0

136.5*

2.29

3

34

3.2

139.3*

8.35

3

33

10

70.6*

3.09

3

66

32

35.4*

4.42

3

83

100

0.8*

0.63

3

100

* - effect was statistically significant

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, Cedarwood Texas oil crude the EL50 for growth rate inhibition (72h-ERL50) was 41 mg/L with a 95% confidence interval between 35 and 48 mg/L.

The EL50 for yield inhibition (72h-EYL50) was 5.0 mg/L with a 95% confidence interval between 4.5 and 5.6 mg/L.
The EL10 for growth rate inhibition (72h-ERL10) was 7.8 mg/L with a 95% confidence interval between 4.8 and 11 mg/L.
The EL10 for yield inhibition (72h-EYL10) was below the loading rate of 1.0 mg/L.
Executive summary:

A full OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata based on the results of a preceding range-finding test.Water Accommodated Fractions (WAFs) were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of 1.0, 3.2, 10, 32 and 100 mg Cedarwood Texas oil crude per litre under static conditions. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. Due to the potential volatile nature of the test item, the exposure was performed with adjusted medium. Test vessels were airtight closed vessels and headspace reduced to minimum. Samples taken from all groups were analysed. A clear increase in measured TOC concentrations was observed in WAFs of 10, 32 and 100 mg/L, indicating proper preparation of WAFs. At the lower WAFs and in the control, no TOC was detected. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. A dose-related increase of inhibition of growth rate and yield was observed at loading rates of 3.2 to 100 mg/L. The highest inhibition was observed in the WAF of 100 mg/L and was 89% for the growth rate and 100% for yield.The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, Cedarwood Texas oil crude the EL50 for growth rate inhibition (72h-ERL50) and yield inhibition (72h-EYL50) was 41 mg/L  and 5.0 mg/L. While the EL10 for growth rate inhibition (72h-ERL10) and yield inhibition (72h-EYL10) was 7.8 mg/L and below the loading rate of 1.0 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
November 30th 2017
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:
Cedarwood Texas oil (crude) is an UVCB substance. Regarding the different solubility of its constituents that make the testing difficult and a known fraction of 93.2%, calculation from the ecotoxicity of the known constituent appears as an acceptable approach.
Principles of method if other than guideline:
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", (1) referenced as Method C.3 of Commission Regulation No. 440/2008 (2) 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 Mode of Action (MOA) in question (non-polar narcosis). It has been validated in an internal publication for MOA 1 (non-polar narcosis) and acute exposure (Bicherel and Thomas, 2014) (3). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004) (4). 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 non-bioavailable 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.

(1) OECD Guideline for testing of chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006; Annex 5 corrected July 28, 2011.
(2) Commission Regulation (EC) No. 761/2009 amending Regulation (EC) No. 440/2008, Part C, C.3.: "Algal Inhibition Test", p. 36-56, Official Journal of the European Union (EN), dated August 24, 2009, L 220:1-94.
(3) Bicherel P and Thomas PC (2014) iSafeRat® WAF calculation method to predict acute aquatic toxicity. Position paper prepared by KREATiS.
(4) OECD (2004) Principles for the validation, for regulatory purposes, of (Quantitative) Structure Activity-Relationship Models, http://www.oecd.org/env/ehs/risk-assessment/oecdquantitativestructure-activityrelationshipsprojectqsars.htm.
GLP compliance:
no
Remarks:
Not required for a calculation based on multiple QSARs method.
Specific details on test material used for the study:
Typical legal entity composition provided by the registrant of the substance, REACH NCS Sesquiterpenes HC / Alc consortium. Composition is as agreed in the Substance Identification Profile version-4 dated 17 Nov 2017. See test material information: Cedarwood Texas oil (crude) (QSAR)

Analytical monitoring:
no
Remarks:
Not relevant for a calculation based on multiple QSARs method
Details on sampling:
Not relevant.
Test organisms (species):
other: green algae
Water media type:
freshwater
Total exposure duration:
72 h
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 66.7 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction
Basis for effect:
growth rate
Remarks on result:
other: Nominal solubility limit/ Calculation based on QSARs
Remarks:
The effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.
Details on results:
The Analytically Measurable Aqueous Phase is maximum 3.88 mg/L of the test item and the Water Accommodated Fraction (WAF) maximum is 66.7 mg/L. Using a loading rate of 66.7 mg/L, i.e. at the maximal solubility of the UVCB in water, and after removal of the non-bioavailable fraction, the remaining solubilised fraction is not sufficient to exert any toxicity effect based growth rate observations. Therefore it is not possible to determine a 72h-ErL50 below the water solubility limit
Validity criteria fulfilled:
yes
Remarks:
QSAR model validated to be compliant with the OECD recommendation for QSAR modelling (OECD, 2004) described within the QMRF.
Conclusions:
The 72h ErL50 (mg test item.L-1) = above water solubility limit ( > 66.7 mg/L)
Executive summary:

A calculation method was used to predict the inhibition of algal growth exposed to the test item Cedarwood Texas oil (crude). 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", (1) referenced as Method C.3 of Commission Regulation No. 440/2008 (2) adapted for testing of a mixture using the WAF method. By using a "known constituents approach" based on non-polar-narcosis MOA-1 QSARs and WAF "adapted water solubilities”, the algae 72h-ErL50 is concluded to be above the WAF estimated at 66.7 mg/L.

Description of key information

A OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata.Water Accommodated Fractions (WAFs) were prepared and used at loading rate of 1.0, 3.2, 10, 32 and 100 mg Cedarwood Texas oil crude per litre under static conditions. Total Organic Carbon (TOC) analysis was performed at the start and at the end of exposure. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. Cedarwood Texas oil crude EL50 for growth rate inhibition (72h-ERL50) and yield inhibition (72h-EYL50) was 41 mg/L  and 5.0 mg/L. While the EL10 for growth rate inhibition (72h-ERL10) and yield inhibition (72h-EYL10) was 7.8 mg/L and below the loading rate of 1.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
41 mg/L
EC10 or NOEC for freshwater algae:
7.8 mg/L

Additional information

An additional study is available to predict the ErL50 for algae by means of QSAR (Kreatis, 2017). In this study the algae 72h-ErL50 was concluded to be above the maximum WAF solubility estimated at 66.7 mg/L. The experimental value is preferred over the predicted value, as the study was fully valid and resulted in a concrete ErL50 and ErL10 value, the QSAR only resulted in a ErL50. In addition, the experimental value is worst-case, but considered of similar order of magnitude.