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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-Nov-16 to 25-Jan-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
December 2015
Deviations:
no
Principles of method if other than guideline:
The test was modified according to the OECD Series on Testing and Assessment Document no. 23 on aquatic toxicity testing of difficult substances (Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000).
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations including control
- Sampling method: For sampling 4.0 mL from the approximate centre of the test vessels were taken at 0, 24 and 72 hours. At the end of each exposure period the replicates were pooled at each concentration before sampling. This occured for all concntrations apart from the 10 % saturated solution as one replicate appeared to have a lighter green appearance and was analysed seperately with the other 2 reminaing replicates pooled for analysis.
- Sample storage conditions before analysis: In a freezer (<-15 °C) until analysis, stability at this storge temperature has been previously confirmed.
Vehicle:
no
Details on test solutions:
The main test was conducted at 1.0, 3.2, 10, 32 and 100 % of a 100 mg/L saturated solution.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was made by adding 100 mg/L of test item to M2-test medium and stirring for 2 days at room temperature. After this period the soluble fraction was siphoned after 1 hour of settling.
- Eluate: N/A
- Differential loading: N/A
- Controls: M2 medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): None used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Tyndall's effect using a laser pen was used to ensure no undissolved particulate was present in the stock solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL-1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 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.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1 medium) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s) in a climate controlled room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not):
Testing conditions are the same, however M2 medium is used in favour of M1 medium.
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Guideline specified
Post exposure observation period:
N/A
Hardness:
24 mg CaCO3/L
Test temperature:
22-23 °C
pH:
7.9-8.1
Dissolved oxygen:
Not reported
Salinity:
N/A
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal Concentrations: 1.0, 3.2, 10, 32 and 100 % of a saturated solution (%SS)
Measured Concentrations (TWA): 0.082, 0.18, 0.65, 0.18, 1.7 and 4.6 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass
- Material, size, headspace, fill volume: 50 mL medium/ medium+test material
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 193.3 to 243.2 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 and 1 extra replicate for sampling purposes, 1 or 2 replicates of each test item concentration without algae.
- No. of vessels per control (replicates): 6 and 1 extra replicate for sampling purposes
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- preparation of dilution water:

M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

- Culture medium different from test medium: Yes, M1 Culture medium:
according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L


- Intervals of water quality measurement: At the beginning and end of the test pH was measured. Temperature was monitored continuously in the control vessel.

OTHER TEST CONDITIONS
- Light intensity and quality: Continuous lighting using TLD-lamps with a light intensity within the range of 82 to 86 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometer with an immersion probe (at 680 nm, pathlength = 20 mm). Algal medium was used as a blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2x
- Range finding study: 1.0, 10 and 100 % of the 100 mg/L saturated solution.
- Test concentrations: 1.0, 3.2, 10, 32 and 100% of a saturated solutions at 100 mg/L
- Results used to determine the conditions for the definitive study: See table 2 and 3
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: Yes, in one vessel at 10 % of the saturated solution. However, algal cells looked healthy and this did not impact the results.
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: N/A
- Any stimulation of growth found in any treatment: Slight stimulation at 0.18 mg/L of test item (-0.5 % inhibition)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid?
- 72h ErC50: 1.3 (95 % confidence interval 1.2-1.4 mg/L)
- 72h ErC10: 0.53 (95 % confidence interval 0.4-0.61 mg/L)
- 72h NOErC: 0.18 mg/L

- 72h EyC50: 0.48 mg/L (95 % confidence interval 0.40-0.57 mg/L)
- 72h EyC10: 0.14 mg/L (95 % confidence interval 0.088-018 mg/L)
- 72h NOEyC: 0.18 mg/L.
- Other: N/A.
Reported statistics and error estimates:
72h ErC10 95% CI: 0.44-0.61 mg/L
72h ErC50 95% CI: 1.0-1.2 mg/L
72h EyC10 95% CI: 0.088-0.18 mg/L
72h EyC50 95% CI: 0.40-0.57 mg/L

Individual growth rates (day-1)

Time

Replicate

 Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
TWA concentration (mg/L)

Control

0.082

0.18

0.65

1.7

4.6

0-24 h

1

1.971

2.024

2.13

1.718

0.727

0

 

2

2.027

2.016

1.921

1.456

0.772

0

 

3

2.031

2.027

2.028

1.817

0.754

0.051

 

4

2.092

 

 

 

 

 

 

5

2.097

 

 

 

 

 

 

6

1.963

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

2.03

2.022

2.027

1.664

0.751

0.017

Std.Dev.:

 

0.0568

0.0058

0.1043

0.1864

0.0226

0.0293

n:

 

6

3

3

3

3

3

CV:

 

2.8

0.3

5.1

11.2

3

173.2

 

 

 

 

 

 

 

0-48 h

1

1.84

1.829

1.905

1.575

0.458

0

 

2

1.913

1.817

1.715

1.175

0.326

0

 

3

1.916

1.803

1.844

1.625

0.366

0.041

 

4

1.895

 

 

 

 

 

 

5

1.906

 

 

 

 

 

 

6

1.877

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

1.891

1.816

1.821

1.458

0.383

0.014

Std.Dev.:

 

0.029

0.0133

0.0973

0.2468

0.0674

0.0236

n:

 

6

3

3

3

3

3

CV:

 

1.5

0.7

5.3

16.9

17.6

173.2

 

 

 

 

 

 

 

0-72 h

1

1.755

1.731

1.815

1.577

0.587

0.011

 

2

1.813

1.735

1.692

1.268

0.37

0.018

 

3

1.831

1.743

1.759

1.615

0.369

0.001

 

4

1.808

 

 

 

 

 

 

5

1.805

 

 

 

 

 

 

6

1.787

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

1.8

1.736

1.756

1.487

0.442

0.01

Std.Dev.:

 

0.0262

0.0059

0.0617

0.1905

0.1259

0.0086

n:

 

6

3

3

3

3

3

CV:

 

1.5

0.3

3.5

12.8

28.5

85.4

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 2%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%.The test fulfils this validity criterion.

Section-by-section growth rate

Time

Replicate

Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
TWA concentration (mg/L)

Control

0.082

0.18

0.65

1.7

4.6

0 - 24 h

1

1.971

2.024

2.13

1.718

0.727

0

 

2

2.027

2.016

1.921

1.456

0.772

0

 

3

2.031

2.027

2.028

1.817

0.754

0.051

 

4

2.092

 

  

 

 

 

 

5

2.097

 

 

 

 

 

 

6

1.963

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

2.03

2.022

2.027

1.664

0.751

0.017

Std.Dev.:

 

0.0568

0.0058

0.1043

0.1864

0.0226

0.0293

n:

 

6

3

3

3

3

3

CV:

 

2.8

0.3

5.1

11.2

3

173.2

 

 

 

 

 

 

 

24 - 48 h

1

1.708

1.634

1.681

1.432

0.188

0

 

2

1.799

1.618

1.508

0.893

-0.119

0

 

3

1.801

1.578

1.659

1.433

-0.023

0.031

 

4

1.697

 

 

 

 

 

 

5

1.716

 

 

 

 

 

 

6

1.791

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

1.752

1.61

1.616

1.253

0.015

0.01

Std.Dev.:

 

0.0496

0.0289

0.0942

0.3114

0.1573

0.0178

n:

 

6

3

3

3

3

3

CV:

 

2.8

1.8

5.8

24.9

1015.4

173.2

 

 

 

 

 

 

 

48 - 72 h

1

1.585

1.535

1.636

1.58

0.847

0.033

 

2

1.613

1.572

1.647

1.453

0.456

0.054

 

3

1.662

1.623

1.59

1.595

0.376

-0.079

 

4

1.634

 

 

 

 

 

 

5

1.602

 

 

 

 

 

 

6

1.607

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

1.617

1.577

1.624

1.543

0.559

0.003

Std.Dev.:

 

0.0271

0.0443

0.0303

0.0779

0.2521

0.0715

n:

 

6

3

3

3

3

3

CV:

 

1.7

2.8

1.9

5

45.1

2297.9

The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 12%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%.The test fulfils this validity criterion.

Individual yields

Time

Replicate

Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal
TWA concentration (mg/L)

Control

0.082

0.18

0.65

1.7

4.6

24 h

1

6.2

6.6

7.4

4.6

1.1

0

 

2

6.6

6.5

5.8

3.3

1.2

0

 

3

6.6

6.6

6.6

5.2

1.1

0.1

 

4

7.1

 

 

 

 

 

 

5

7.1

 

 

 

 

 

 

6

6.1

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

6.6

6.6

6.6

4.3

1.1

0

Std.Dev.:

 

0.43

0.04

0.79

0.95

0.05

0.03

n:

 

6

3

3

3

3

3

CV:

 

6.5

0.7

12

22

4.3

173.2

 

 

 

 

 

 

 

48 h

1

38.6

37.8

44.2

22.3

1.5

0

 

2

44.9

36.8

29.9

9.5

0.9

0

 

3

45.2

35.8

39

24.8

1.1

0.1

 

4

43.2

 

 

 

 

 

 

5

44.3

 

 

 

 

 

 

6

41.7

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

43

36.8

37.7

18.9

1.2

0

Std.Dev.:

 

2.48

1.01

7.25

8.23

0.3

0.05

n:

 

6

3

3

3

3

3

CV:

 

5.8

2.7

19.2

43.6

25.6

173.2

 

 

 

 

 

 

 

72 h

1

192.3

179.1

230.9

112.3

4.8

0

 

2

229.2

181.2

159.2

43.8

2

0.1

 

3

242.2

185.5

194.9

126.2

2

0

 

4

225.7

  

 

 

 

 

 

5

223.7

 

 

 

 

 

 

6

211.9

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean:

 

220.8

181.9

195

94.1

3

0

Std.Dev.:

 

17.02

3.25

35.83

44.09

1.62

0.03

n:

 

6

3

3

3

3

3

CV:

 

7.7

1.8

18.4

46.8

54.6

85.9


pH levels recorded during the final test

TWA conc. (mg/L)

pH

t=0h

t=72h

Control

8.1

8.1

0.082

8.1

8.0

0.18

8.0

8.0

0.65

8.0

8.0

1.7

8.0

7.9

4.6

7.9

7.9

 

Validity criteria fulfilled:
yes
Conclusions:
The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.0 to 1.2 mg/L.
The EC10 for growth rate inhibition (72h-ERC10) was 0.53 mg/L with a 95% confidence interval ranging from 0.44 to 0.61 mg/L.
The 72h-NOEC for growth rate inhibition yield inhibition was 0.18 mg/L.

The EC50 for yield inhibition (72h-EYC50) was 0.48 mg/L with a 95% confidence interval ranging from 0.40 to 0.57 mg/L.
The EC10 for yield inhibition (72h-EYC10) was 0.14 mg/L with a 95% confidence interval ranging from 0.088 to 0.18 mg/L.
The 72h-NOEC for yield inhibition was 0.18 mg/L.
Executive summary:

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266, 2015 and the OECD series on testing and assessment number 23, 2000.

The batch of Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal tested was a colourless liquid with a purity of 99.6% (sum of 2 diastereomers). The test item was not completely soluble in test medium at the loading rate initially prepared.

A final test was performed based on the results of a preceding combined limit/range-finding test. An initial stock solution at 100 mg/L was prepared by adding measured amounts of the test item to test medium and mixing for 2 days to achieve an equilibrated concentration of dissolved constituents in the aqueous phase. Following cessation of mixing, and a 1 hour period of settling, the saturated solution (SS) was siphoned out andchecked for tyndall effect, using a Laser pen. The clear and colourless SS was used as the highest test concentration. Lower concentrationswere obtainedvias erial dilution of the SS in test medium.

Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100% of the SS. The initial algal cell density was 104cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.23, 0.66, 3.2, 8.8 and 31 mg/L at 1.0, 3.2, 10, 32 and 100% of the SS at 100 mg/L, respectively. All concentrations decreased to 15-51% of initial during the first 24 hours of exposure and further to concentrations below the limit of detection (LOD=0.013 mg/L) at the end of the test period. Based on these results the time weighted average concentrations were calculated to be 0.082, 0.18, 0.65, 1.7 and 4.6 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

 

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.18

0.53

0.69

1.1

lower 95%-cl

 

0.44

0.60

1.0

upper 95%-cl

 

0.61

0.76

1.2

Yield

Value

0.18

0.14

0.21

0.48

lower95%-cl

 

0.088

0.15

0.40

upper 95%-cl

 

0.18

0.26

0.57

 

Description of key information

72 h ErC50 (Pseudokirchneriella subcapitata) = 1.1 mg/L, OECD 201 Freshwater alga and cyanobacteria, growth rate inhibition test, (Anon., 2017).

72 h ErC10 (Pseudokirchneriella subcapitata) = 0.53 mg/L, OECD 201 Freshwater alga and cyanobacteria, growth rate inhibition test, (Anon., 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.1 mg/L
EC10 or NOEC for freshwater algae:
0.53 mg/L

Additional information

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266, 2015 and the OECD series on testing and assessment number 23, 2000.

The batch of Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal tested was a colourless liquid with a purity of 99.6% (sum of 2 diastereomers). The test item was not completely soluble in test medium at the loading rate initially prepared.

A final test was performed based on the results of a preceding combined limit/range-finding test. An initial stock solution at 100 mg/L was prepared by adding measured amounts of the test item to test medium and mixing for 2 days to achieve an equilibrated concentration of dissolved constituents in the aqueous phase. Following cessation of mixing, and a 1 hour period of settling, the saturated solution (SS) was siphoned out andchecked for tyndall effect, using a Laser pen. The clear and colourless SS was used as the highest test concentration. Lower concentrationswere obtainedvias erial dilution of the SS in test medium.

Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100% of the SS. The initial algal cell density was 104cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.23, 0.66, 3.2, 8.8 and 31 mg/L at 1.0, 3.2, 10, 32 and 100% of the SS at 100 mg/L, respectively. All concentrations decreased to 15-51% of initial during the first 24 hours of exposure and further to concentrations below the limit of detection (LOD=0.013 mg/L) at the end of the test period. Based on these results the time weighted average concentrations were calculated to be 0.082, 0.18, 0.65, 1.7 and 4.6 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

 

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.18

0.53

0.69

1.1

lower 95%-cl

 

0.44

0.60

1.0

upper 95%-cl

 

0.61

0.76

1.2

Yield

Value

0.18

0.14

0.21

0.48

lower95%-cl

 

0.088

0.15

0.40

upper 95%-cl

 

0.18

0.26

0.57