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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 2015-02-16 to 2015-02-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 201 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on June 03-05, 2013 / signed on November 05, 2013
Specific details on test material used for the study:
- Water solubility in pure water: isomer 1 = 125 µg/L; isomer 2 = 285 µg/L
- Water solubility in M4 medium: isomer 1 = 115 µg/L; isomer 2 = 261 µg/L
(determined at Dr. U.NOACK-LABORATORIEN, Noack Lab-ID: 131029FG/CWE15836)
Analytical monitoring:
yes
Details on sampling:
The saturated solution and the control were analytically verified via GC-MS at the beginning and the end of exposure. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, separate replicates were prepared with algae at the beginning of exposure and incubated under test conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The saturated solution with a nominal loading of 2.00 mg/L was prepared once with dilution water. The saturated solution was stirred for 24 hours (1100 rpm) with a magnetic stirrer.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE/m2/s for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
Dilution water medium has a nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
21.8-23.5 °C (22.7 °C)
pH:
8.20 - 9.49
Dissolved oxygen:
None
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Saturated solution with a nominal loading rate of 2.00 mg/L was tested as a limit test loading.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 119 mL, with aluminium tops with PTFE seals
- Test volume: 119 mL
- Initial cells density: Nominal: approximately 1 x 10^4 cells/mL; Current: 7085 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Application: Application was carried out by adding appropriate volumes of the saturated solution to the test replicates.
- Incubation: The flasks were positioned randomly and repositioned daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the OECD guideline 201

OTHER TEST CONDITIONS
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 100 rpm.
- Photoperiod: 24 hours/day light
- Light intensity and quality: 84.2 to 94.1 (90.4) µE/m2/s
- Light homogeneity: Within ± 15 % over incubation area

EFFECT PARAMETERS MEASURED:
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the range finding and in the definitive test at the nominal concentration level of 2.00 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.
Physical Chemical Parameters:
- The pH-value at the beginning and end of the test was measured from pooled replicates per concentration and control after measurement of the cell density, respectively. The room temperature was measured continuously. The light intensity was measured prior to the test start.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP preliminary range finding test, three nominal test concentrations of 20, 200 and 2000 µg/L were tested. Inhibition of growth rate was -3, -3 and 3 % at test concentrations of 20, 200 and 2000 µg/L after 72 h. Respective inhibitions of yield were -10, -12 and 13 %.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > solubility limit
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > solubility limit
Details on results:
Biological data:
Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in saturated solution and control. All effect values are given based on geometric mean measured test item concentration.

Measured Exposure Concentrations during the Definitive Test:
The concentrations of the test item was determined at the start of exposure (0 h) and at the end of the exposure in the test item loading level and the control via GC-MS. The measured concentration of the test item at the start of exposure was 101 % of the nominal value. At the end of exposure the measured concentration was 50 % of the nominal value. All effect values given were based on geometric mean measured concentrations of the test item.
Results with reference substance (positive control):
- Results with reference substance valid
- The ErC50 value based on Growth Rate Inhibition: 0.749 mg/L (with Headspace) and 0.774 (without Headspace)
- The EyC50 value based on Yield Inhibition: 0.282 mg/L (with Headspace) and 0.502 (without Headspace)
Reported statistics and error estimates:
EC-values and statistical analyses: EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (limit test design).

NOEC, LOEC and statistical analyses: The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. Normality Test failed first for yield data but was passed after a square root data transformation.

Table 6.1.5/1: Cell Densities (Geometric mean measured test item concentrations)

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

1.42

1

7085

18971

139626

559075

2

7085

18253

118265

552468

3

7085

15189

135934

562461

4

7085

11989

132787

619731

5

7085

22470

144241

539122

6

7085

22686

153902

565848

Mean

7085

18260

137459

566451

Control

1

7085

28403

176033

592474

2

7085

22440

157873

626139

3

7085

26600

179074

559772

6

7085

28416

180250

631418

7

7085

24060

185840

643934

8

7085

32596

201790

770260

Mean

7085

27086

180143

637333

Table 6.1.5/2: Evaluation after 72 hours

 

Geometric mean measured test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1.42

1

 

1.46

3

 

551990

12

2

 

1.45

3

 

545383

13

3

 

1.46

3

 

555376

12

4

 

1.49

1

 

612646

3

5

 

1.44

4

 

532037

16

6

 

1.46

3

 

558763

11

Mean

(+)

1.46

3

(+)

559366

11

Control

1

 

1.48

 

 

585389

 

2

 

1.49

 

 

619054

 

3

 

1.46

 

 

552687

 

4

 

1.50

 

 

624333

 

5

 

1.50

 

 

636849

 

6

 

1.56

 

 

763175

 

Mean

 

1.50

 

 

630248

 

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 


Table 6.1.5/3:Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

 

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.39

1.82

1.21

1.48

0.314

21.3

24.0

2

1.15

1.95

1.38

1.49

0.412

27.5

3

1.32

1.91

1.14

1.46

0.401

27.5

4

1.39

1.85

1.25

1.50

0.311

20.8

5

1.22

2.04

1.24

1.50

0.469

31.2

6

1.53

1.82

1.34

1.56

0.244

15.6

NA

Mean

1.50

NA

 

SD ±

0.04

 

CV [%]

2.40

 

SD = Standard deviation          CV = Coefficient of variation

 

Table 6.1.5/4: Environmental Conditions

Geometric mean measured test item concentration

pH-value

[mg/L]

Start; 0 hours

End; 72 hours

1.42

8.20

9.49

Control

8.21

9.47

Room temperature [°C]:

min.: 21.8

max.: 23.5

mean value: 22.7

 

Light intensity
[µE∙m-2∙s-1]

n = 20

mean value: 90.4

range of the measured values: 84.2 to 94.1 equalling -6.81 to 4.15 %

CV [%]: 3.11

CV = Coefficient of variation                 n = number of measuring points

 

The study meets the validity criteria of the guideline:

 

Validity Criteria

Validity Criterium

Required

This study

Increase of the cell growth in the control cultures

Exponentially > 16-fold corresponding to a specific growth rate of 0.92 day-1

90-fold
(specific growth rate 1.50 day-1)

Mean coefficients of variation for section-by-section specific growth rates (days           0-1, 1-2 and 2-3) in the control cultures

< 35 %

24.0 %

Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures

< 7 %

2.40 %

Validity criteria fulfilled:
yes
Conclusions:
Only 3% inhibition of the growth rate was observed at the geometric mean measured concentration of 1.42 mg/L. Therefore, the 72h-ErC10 and 72h-ErC50 were determined to be greater than this value, greater than the solubility limit.
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at the nominalloading level of 2.00 mg/Lfor 72 h. The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Six replicates were tested for the test item concentration and for the control, respectively. The environmental conditions were within the acceptable limits.

 

The concentration of the saturated solution of the test item and the control were analytically verified via GC-MS at the start and the end of exposure. The recovery rate ofthe test item at the start was 101 %, at the test end it was determined to be 50 %. Therefore, all effect values were based on the geometric mean measured test item loading rate (1.42 mg/L).

 

Under the test conditions, only 3% inhibition of the growth rate was observed at the geometric mean measured concentration of 1.42 mg/L. Therefore, the 72h-ErC10 and 72h-ErC50 were determined to be greater than this value, greater than the solubility limit.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances (stereoisomers) and comparable properties related to the target endpoints.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
See attached document in Iuclid section 13

4. DATA MATRIX
See attached document in Iuclid section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Remarks:
Read-Across justification document
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > solubility limit
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > solubility limit
Validity criteria fulfilled:
yes
Conclusions:
According to the experimental study performed on the source substance, only 3% inhibition of the growth rate was observed at the geometric mean measured concentration of 1.42 mg/L. Therefore, the 72h-ErC10 and 72h-ErC50 were determined to be greater than this value, greater than the solubility limit.
Executive summary:

No experimental study is available on the target substance to assess the toxicity of the registered substance to aquatic algae. Therefore, good quality data for a related source substance (Reaction mass of (3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and (3S*)-1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]hexan-3-ol (EC# 942-425-2)) have been read-across for this endpoint. The target and the source substance are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the target/registered substance are constituents of the source substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantionmers). 


The algal growth inhibition study, performed on the source substance, was conducted according to OECD Guideline 201 and in compliance with GLP. Freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at the nominal loading level of 2.00 mg/Lfor 72 h. The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Six replicates were tested for the test item concentration and for the control, respectively. The environmental conditions were within the acceptable limits.  


The concentration of the saturated solution of the test item and the control were analytically verified via GC-MS at the start and the end of exposure. The recovery rate ofthe test item at the start was 101 %, at the test end it was determined to be 50 %. Therefore, all effect values were based on the geometric mean measured test item loading rate (1.42 mg/L).  


Under the test conditions, only 3% inhibition of the growth rate was observed at the geometric mean measured concentration of 1.42 mg/L. Therefore, the 72h-ErC10 and 72h-ErC50 were determined to be greater than this value, greater than the solubility limit.


Therefore, based on the considerations above, it can be concluded that the result of the algae study conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VII, 9.1.4 of the REACH regulation.

Description of key information

Read-across, OECD Guideline 201, GLP, key study, validity 2:

No effects were observed on Pseudokirchneriella subcapitata up to the solubility limit of the test substance.

Key value for chemical safety assessment

Additional information

To assess the toxicity of the registered (target) substance to aquatic algae, one experimental study is available on a source substance.


This valid GLP study, assessed as a key study, was performed on the source substance, Reaction mass of (3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and (3S*)-1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]hexan-3-ol (EC# 942-425-2), according to OECD Guideline 201. The target and the source substance are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the target/registered substance are constituents of the source substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantionmers). 


 


In this study, the unicellular freshwater green alga Pseudokirchneriella subcapitata was exposed to a saturated solution of the test substance with a nominal loading level of 2 mg/L and a control, under static conditions, over a period of 72 hours. The concentrations of the saturation solution of the test substance and the control were analytically verified via GC-MS at the start and at the end of the exposure. The recovery rate of the test substance at the start was 101% and at the test end it was determined to be 50%. Therefore, all effects values were based on geometric mean measured test substance loading rate. According to the results of this study, only 3% inhibition of the growth rate was observed at the geometric mean measured concentration of 1.42 mg/L. Therefore, the 72h-ErC10 and 72h-ErC50 were determined to be greater than this value, greater than the solubility limit of the test substance.


 


This source substance is considered adequate for read-across purposes based on structural similarity (stereoisomers), on similar basic physico-chemical properties and similar environmental profile between the registered substance and the analogue material used (see IUCLID section 13 for justification).