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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 201 guideline without deviation. The substance was a multi-constiuent of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
July 2011
Deviations:
no
Remarks:
A WAF design is used in accordance with in accordance with the OECD Series on testing and assessment No. 23
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
- Principle of test: Water accomOdated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- Short description of test conditions: Each concentration is prepared sepArately, whereby the test item is added to the medium at a specific loading rate. The material is left until fully dissolved (or as dissolved as possible), then particulate matter is left to settle out of the water column. The remaining substance in the water column of the sample is transferred to the test vessels. This is done at each concentration. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Tyndall's effect is monitored to ensure minimal particulate matter is transferred to the test system. Further, chemical analysis is conducted to confirm that the correct loading has occurred, presence of the test item and to confirm there is a series of varying concentrations.
- although the study was conducted in accordance with the OECD guideline 203 (Fish acute toxicity test, 1992) as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  However, during study performance a deviation to the study design was noted. Here only the 100 mg/L test loading rate was prepared in accordance with the WAF strategy. All other concentrations were made by serial dilution, even though weighing will have been possible. As no effects were seen at the limit dose which was not prepared by serial dilution, i.e. in accordance with OECD series on testing and assessment no. 23, the study is considered valid and the deviation has not impacted the integrity of the study as a limit test.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: N/D

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All tested concentrations were analysed at 0 and 72 hours, the concentraions were 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L.
- Sampling method: direct from vessel
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accomadated fractions were used as per the OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002). Each concentration was separately prepared. The prerequisite amount of test item was added to the test vessel containing the test medium 1000 mL of test medium for the concentrations of 100, 31.3, 9.77 and 3.05 mg/L stock loading rates or 2000 mL for the 0.954 mg/L stock loading rate. The final loading rate of 0.298 mg/L was made by diluting stock solution S5 with test medium without algae to give the required test item loading rates, this was due to the fact that the weighing of such small amounts was not possible or practicable, and therefore did not allow for direct addition of the concentration to the test system for the lowest concentration. This is in accordance with OECD Series on Testing and Assessment No. 23. The stocks were then stirred for 24 h in the dark at room temperature. The stock solutions were then allowed to settle to allow non-dissolved test item to sediment or float to the surface for 1 hour. After this the necessary volumes for the test were withdrawn via a Teflon tube from the middle level of the stock solution. Algae were added to each solution individually resulting in nominal cell densities of 0.5 × 10^4 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel. See table 1 for details on test preparations.
- Eluate: N/A
- Differential loading: N/A
- Controls: AAP medium only (OECD 201, 2011)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): A 1 hour settling time was chosen as no Tyndall's affect was seen in the lower 5 concentrations (i.e. undissolved material was not detected). At 100 mg/L there was a mild Tyndall effect however, a pre-test showed no improvement or reduction in the Tyndall effect and thus undissolved material if the 100 mg/L concentration was left to settle for a further 48 hours. Therefore, 1 hour settling was seen as optimum for all concentrations and used for the definitive test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.
Culture conditions were as follows:
Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1)
Temperature: 21 - 24 °C
Culture flasks: 100 mL Erlenmeyer flasks
CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Cells from this semi-continuous liquid stock culture were used for the test.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Remarks:
AAP-Medium (according to Annex 3 of OECD 201, 2011)
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
OECD 201 guideline duration.
Post exposure observation period:
none
Hardness:
N/D
Test temperature:
22.1-22.8
pH:
7.38-9.76
Dissolved oxygen:
N/D
Salinity:
N/D
Conductivity:
N/D
Nominal and measured concentrations:
Nominal loading rates were, 100 mg/L, 31.3 mg/L, 9.77 mg/L, 3.05 mg/L, 0.954 and 0.298 mg/L (these concentrations are based on test substance added minus water content, as water is not the driver for any observed toxicity). Measured concentrations were 85 to 115 % of nominal. Nominal loading rates were used as analysis was only for verification of correct loading rates as a WAF design was used.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with 50 mL medium-algae, or medium-algae-test item.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 0.5 x 10^4
- Control end cells density: 65.18 x 10^4
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): no vehicle was used and no vehicle controls were required.

GROWTH MEDIUM
- Standard medium used: yes (AAP medium, OECD 201)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory prepared.
- Total organic carbon: not relevant/required
- Particulate matter: not relevant/required
- Metals: not relevant
- Pesticides: not relevant
- Chlorine: not relevant/required
- Alkalinity: not relevant/required
- Ca/mg ratio: not relevant/required
- Conductivity: not relevant/required
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72.

OTHER TEST CONDITIONS
- Sterile test conditions: not required
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.
- Salinity (for marine algae): not relevant

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter, calibrated using cell counter Neubauer chamber.
- Chlorophyll measurement: none
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: 5 concentrations used in accordance with OECD guideline 201
- Range finding study: Yes, conducted at 0, 7.5 and 75 mg/L.
- Test concentrations: 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
- Results used to determine the conditions for the definitive study: no effect at 7.5 or 75 mg/L were observed in the range finding test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate was used as the positive control.
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
3.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
5.08 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
1.39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
1.95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
3.74 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
9.77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.954 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
3.05 mg/L
Nominal / measured:
nominal
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: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: -
- Any stimulation of growth found in any treatment: there was a mild increase in growth rate between 0.298 to 0.954 mg/L, but within the rnage of normal biogical variation (< 10 %).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: loading rates were used.
- Effect concentrations exceeding solubility of substance in test medium: some particulate matter was observed at the highest concntration of 100 mg/L. However, this is the limit concentration as advised by the OECD 201 guideline and at both 31.3 mg/L and 100 mg/L 100 % inhibition was seen. This does not efect the integrity of the result and is in line with the OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Doc No. 23, 2002)

Furthermore, the data on the measured analyte showed presence of the test material in a series of conctrations of which biological effect could be assessed, appropriiate for data analysis using a WAF apporach (Table 8).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.23 mg/L (growth rate)
- Other: NOEC 0.320 mg/L, LOEC 0.800 mg/L
Reported statistics and error estimates:
The statistical evaluation for the 72 hour period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for growth rate and yield).

Table 3 Individual cell number counts at timepoint x

Loading rate

Cell numbers [104/mL]

Yield

[mg/L]

0 h

24 h

48 h

72 h

0 h – 72 h

0.69

2.10

13.22

56.77

56.08

0.59

2.31

12.05

51.49

50.90

Control

0.52

1.75

10.80

46.81

46.29

0.63

1.75

10.27

53.41

52.78

0.67

1.70

10.16

50.81

50.14

0.58

1.78

11.11

48.23

47.65

Mean

0.611)

1.90

11.27

51.25

50.64

0.298

0.61

2.10

11.54

47.96

47.35

0.61

1.82

9.69

54.32

53.71

0.61

2.07

13.10

57.18

56.57

Mean

0.61

2.00

11.44

53.15

52.54

0.61

1.73

12.64

59.36

58.75

0.954

0.61

1.99

10.23

59.25

58.64

0.61

2.19

10.46

51.42

50.81

Mean

0.61

1.97

11.11

56.68

56.07

0.61

1.55

6.42

31.16

30.55

3.05

0.61

0.81

4.98

23.98

23.37

0.61

1.44

4.51

21.06

20.45

Mean

0.61

1.27

5.30

25.40

24.79

0.61

0.60

1.54

5.88

5.27

9.77

0.61

0.81

2.56

8.85

8.24

0.61

0.64

3.29

11.53

10.92

Mean

0.61

0.68

2.46

8.75

8.14

0.61

0.19

0.15

0.08

-0.53

31.3

0.61

0.22

0.14

0.34

-0.27

0.61

0.32

0.17

0.32

-0.29

Mean

0.61

0.24

0.15

0.25

-0.36

0.61

0.03

0.00

0.00

-0.61

100

0.61

0.09

0.03

0.00

-0.61

0.61

0.04

0.04

0.01

-0.60

Mean

0.61

0.05

0.02

0.00

-0.61

Table 4 Inhibition of growth rate %

Loading rate

% Inhibition of growth rate

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

1.3

-1.4

0.4

-21.0

-3.6

-0.9

Control

-7.6

-4.2

-1.6

9.4

4.1

-0.3

17.4

6.6

2.2

0.6

-1.4

0.2

Mean

0.0

0.0

0.0

 

-9.6

-1.0

1.4

0.298

3.1

5.0

-1.4

 

-8.4

-5.4

-2.5

 

-5.0

-0.5

-0.81)

7.6

-4.1

-3.4

0.954

-4.9

3.1

-3.3

-13.4

2.4

-0.1

Mean

-3.6

0.5

-2.32)

17.3

19.1

11.2

3.05

74.9

27.9

17.1

23.8

31.3

20.0

Mean

38.7

26.1

16.1

101.5

68.2

48.8

9.77

74.9

50.7

39.6

95.7

42.1

33.6

Mean

90.7

53.7

40.7

203.4

148.2

145.9

31.3

190.4

150.6

113.2

157.2

143.9

114.6

Mean

183.7

147.6

124.6 *3)

367.1

100.0

100.0

100

269.7

203.5

100.0

341.6

193.6

192.8

Mean

326.1

165.7

130.9 *1)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

3)Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 5 Inhibition % of yield

Loading rate

% Inhibition of yield

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

-9.3

-17.5

-10.7

-33.3

-7.5

-0.5

Control

4.7

3.6

8.6

13.2

9.6

-4.2

20.2

11

1.0

7

1.2

5.9

Mean

0.4

0.1

0.0

 

-15.5

-2.5

6.5

0.298

6.2

14.8

-6.1

 

-13.2

-17.2

-11.7

 

-7.5

-1.6

-3.81)

13.2

-12.9

-16

0.954

-7

9.8

-15.8

-22.5

7.6

-0.3

Mean

-5.4

1.5

-10.72)

27.1

45.5

39.7

3.05

84.5

59

53.9

35.7

63.4

59.6

Mean

49.1

56.0

51.1 *

100.8

91.3

89.6

9.77

84.5

81.7

83.7

97.7

74.9

78.4

Mean

94.6

82.6

83.9 *

132.6

104.3

101.0

31.3

130.2

104.4

100.5

122.5

104.1

100.6

Mean

128.7

104.3

100.7 *3)

145.0

105.7

101.2

100

140.3

105.4

101.2

144.2

105.3

101.2

Mean

143.2

105.5

101.2 *1)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

3)Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 6 pH of the test system

Loading rate

t = 0 h

t = 72 h

[mg/L]

Control

7.50

8.69

0.298

7.48

8.63

0.954

7.38

8.45

3.05

7.38

8.26

9.77

7.45

8.11

31.3

8.62

7.94

100

9.76 

7.98

Table 7 Temperature in the test system

Time

Temperature [°C]

[h]

min

max

0

22.6

24

22.1

22.8

48

22.2

22.8

72

22.2

22.8

Table 8 measured concentration of the analyte dibutylamine

Test item nominal
(loading rate)
[mg/L]

Sampling

Test item found

[mg/L]

% of nominal

Control

0 h fresh

<LOD

-

72 h aged

<LOD

-

0.298

0 h fresh

0.155

52

72 h aged

< LOD

-

0.954

0 h fresh

0.480

50

72 h aged

< LOD

-

3.05

0 h fresh

2.55

84

72 h aged

< LOD

-

9.77

0 h fresh

8.76

90

72 h aged

0.0236

0.2

31.3

0 h fresh

25.9

83

72 h aged

19.5

62

100

0 h fresh

90.3

90

72 h aged

92.6

93

Table1: pH measurements during the test

Loading rate

t = 0 h

t = 72 h

[mg/L]

Control

7.50

8.69

0.298

7.48

8.63

0.954

7.38

8.45

3.05

7.38

8.26

9.77

7.45

8.11

31.3

8.62

7.94

100

9.76

7.98

Validity criteria fulfilled:
yes
Conclusions:
A test was conducted to determine the effects of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine on the growth of the single cell green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.

The LOEL for growth was determined to be 9.77 mg/L (nominal), the corresponding NOEL was set at 3.05 mg/L (nominal).
The LOEL for yield was determined to be 3.05 mg/L (nominal), the corresponding NOEL was set at 0.954 mg/L (nominal).
The EL10-value for growth rate (ErL10) was determined to be 3.24 mg/L (nominal). The EL10-value for yield (EyL10) was 1.39 mg/L (nominal).
The EL20-value for growth rate (ErL20) was determined to be 5.08 mg/L (nominal). The EL20-value for yield (EyL20) was 1.95 mg/L (nominal).
The EL50-value for growth rate (ErL50) was determined to be 10.0 mg/L (nominal). The EL50-value for yield (EyL50) was 3.74 mg/L (nominal).
Executive summary:

The objective of this study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate (EL10, 20, 50), were estimated.

The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L. WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 1 hour. One concentration (the lowest) was prepared by dilution as it was not possible to weigh the required small amounts to allow for direct addition. Some particles were still present in the upper two concentrations as indicated by the Tyndall's effect but this did not impact the integrity of the study. Furthermore, longer settling periods did not reduce the presence of particulate in these vessels. Water quality parameters were measured at the start and end of the test or daily. Analytical confirmation of the test substance was performed at each concentration at 0 and 72 hours. All water quality parameters and study validity criteria were met as per OECD guideline 201. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

The LOEL for growth was determined to be 9.77 mg/L (nominal), the corresponding NOEL was set at 3.05 mg/L (nominal).

The LOEL for yield was determined to be 3.05 mg/L (nominal), the corresponding NOEL was set at 0.954 mg/L (nominal).

The EL10-value for growth rate (ErL10) was determined to be 3.24mg/L (nominal). The EL10-value for yield (EyL10) was1.39 mg/L (nominal).

The EL20-value for growth rate (ErL20) was determined to be 5.08mg/L (nominal). The EL20-value for yield (EyL20) was1.95 mg/L (nominal).

The EL50-value for growth rate (ErL50) was determined to be 10.0mg/L (nominal). The EL50-value for yield (EyL50) was3.74 mg/L (nominal).

Description of key information

72h EL50 (growth rate) 10 mg/L, 72h EL10 (growth rate) 3.24 mg/L (Pseudokirchnerella subcapitata); OECD 201; Schuster A.-K. 2018,

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L
EC10 or NOEC for freshwater algae:
3.24 mg/L

Additional information

A single key study was conducted to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate (EL10, 20, 50), were estimated.

The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L. WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 1 hour. One concentration (the lowest) was prepared by dilution as it was not possible to weigh the required small amounts to allow for direct addition. Some particles were still present in the upper two concentrations as indicated by the Tyndall's effect but this did not impact the integrity of the study. Furthermore, longer settling periods did not reduce the presence of particulate in these vessels. Water quality parameters were measured at the start and end of the test or daily. Analytical confirmation of the test substance was performed at each concentration at 0 and 72 hours. All water quality parameters and study validity criteria were met as per OECD guideline 201. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

The LOEL for growth was determined to be 9.77 mg/L (nominal), the corresponding NOEL was set at 3.05 mg/L (nominal).

The LOEL for yield was determined to be 3.05 mg/L (nominal), the corresponding NOEL was set at 0.954 mg/L (nominal).

The EL10-value for growth rate (ErL10) was determined to be 3.24mg/L (nominal). The EL10-value for yield (EyL10) was1.39 mg/L (nominal).

The EL20-value for growth rate (ErL20) was determined to be 5.08mg/L (nominal). The EL20-value for yield (EyL20) was1.95 mg/L (nominal).

The EL50-value for growth rate (ErL50) was determined to be 10.0mg/L (nominal). The EL50-value for yield (EyL50) was3.74 mg/L (nominal).