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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:
20 - 23 July 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study conducted under GLP meeting guideline validity criteria. Substance was not detectable after 72 hours due to low solubility of the test item and degradation/transformation/flocculation in the test system. This was deemed acceptable and unavoidable. The presence of EDTA may have also impacted chemical availability to the organism.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Annex 5 corrected: 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline No. 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
September 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark.
- Stability under test conditions: See analytical results section.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in the test media was determined to be 0.960 mg/L. See analytical results section for stability information.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Seperate water accomodated fractions ( WAF's) prepared for each dose rate (nominal dose rate: 3.1, 6.3, 13, 25, 50 and 100 mg/L). The test sample and medium were mixed with a magnetic stirrer for 48 h. The suspension was then filtered with a membrane filter (GV, 0.22 µm, Merck Millipore).
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: See above.
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Seperate water accomodated fractions ( WAF's) prepared for each dose rate.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Individual WAF's prepared at each nominal dose rate; 3.1, 6.3, 13, 25, 50 and 100 mg/L

- Sampling method:
The test item is composed of CaO, MnO2 and TiO2. Each metal ion was individually quantified, and the concentration of metals deriving the test item were calculated to their oxidation number. The sum of these concentrations was defined as the test item concentration.

In the 100 mg/L dose rate test group, the concentration of the test item was measured at the start of exposure, 24, 48 and 72 h after the start of exposure. Other dose groups were measured at the start and end of the exposure period (i.e. 0 and 72 h).

Subsamples were taken (100 mL) were taken from all test solutions at 0 h. 100 mL was taken from sacrificial tests flasks at 24 and 48 h timepoints in the 100 mg/L test group. 90 mL was taken from each test flask and pooled for each respective test group at 72 h.

- Sample storage conditions before analysis:
Not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual WAF solutions prepared for each of the nominal loading rate. Weighed amounts of test item were added to test medium to prepare nominal loading rates of 3.1, 6.3, 13, 25, 50 and 100 mg/L. Solutions were stirred using a magnetic stirrer for 48 h. The suspensions were then filtered through a membrane filter (GV, 0.22 µm pore size, Merck Millipore) by suction. The filrate was used as the representative test solution and was divided into separate test replicates.
- Eluate: Algae media was prepared in purified water in accordance with OECD 201.
- Controls: Algae media only, treated in the same manner as the test solutions.
- 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)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, at the start of the experiment all solutions appeared clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata / green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): Laboratory culture
- Age of inoculum (at test initiation): 72 h - pre-culture incubated under test conditions for 3 days prior to test initiation.
- Method of cultivation: Passage cultured under sterile conditions in the laboratory. The pre-culture was incubated under the same conditions as the test for 3 days prior to test initiation.

ACCLIMATION
- Acclimation period: The pre-culture was incubated under the same conditions as the test for 3 days prior to test initiation.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
-
Test temperature:
22.2-22.5 ºC
pH:
7.8-7.9
Dissolved oxygen:
-
Salinity:
-
Conductivity:
-
Nominal and measured concentrations:
Nominal: 3.1, 6.3, 13, 25, 50 and 100 mg/L
Measured: Test item was only detected in the 100 mg/L nominal test solution through the course of the study. Based on CaO, MnO2 and TiO2 concentrations the geometric mean concentration for the 100 mg/L nominal test group was 0.200 mg/L. Using the geometric spacing factor between concentrations the 3.1, 6.3, 13, 25 and 50 mg/L (nominal) test concentrations were estimated to be 0.00625, 0.0125, 0.0250, 0.0500 and 0.100 mg/L, respectively, based on the geometric mean measured concentrationof the top concentration group i.e. 0.200 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Not reported
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: Filled with 100 mL test solution. Size and type of vessel not reported.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 0.75E4
- Control end cells density:
- No. of vessels per concentration (replicates): 3 (4 for 100 mg/L as additional sacrificial vessel was prepared for analysis at 24 and 48 h)
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): No

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory prepared with purified water in accordance with OECD 201.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH = test start and end. Temperature and light intensity = test start, 24, 48 h and test end.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH:
- Photoperiod: Continuous
- Light intensity and quality: 90 µmol/m2/sec in the wavelength 400-700 nm (actual: 86-89 µmol/m2/sec)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell growth measured every 24 h
- Determination of cell concentrations: Electonic particle counter (Coulter Z2, Beckman Coulter Inc., Instrument No. CC-006)
- Chlorophyll measurement: Not measured
- Other: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.1, 0.57, 3.2, 18 and 100 mg/L nominal
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is routinely tested. The results from the previous positive control test with potassium dichromate were within the normal ranges for this reference item.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Cells slightly enlarged/ bloated at 72 h in the 50 and 100 mg/L (nominal) test groups.
- Unusual cell shape: As above.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any stimulation of growth found in any treatment: No
- 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: EL50 and NOELR both exceed the solubility of the test item in the test solution. Solubility of the test item was established as up to 0.960 mg/L in the preliminary study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.3 mg/L
- Other: No
Reported statistics and error estimates:
EL50 / EC50 (growth rate): These were estimated as greater than the highest tested concentrations since 50 % inhibition was not observed at the highest tested concentration.
NOELR / NOEC (growth rate): Bartletts test was done to determine the homogeneirty of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test was used to determine the significant difference between the control and the exposure levels. The statistical analysis was conducted using Microsoft Excel.

Table 1 Test solution pH values      

 

Nominal loading concentration

(mg/L)

pH

Test start

Test end

Control

7.9

7.8

3.1

7.9

7.8

6.3

7.9

7.8

13

7.9

7.8

25

7.9

7.8

50

7.9

7.8

100

7.9

7.8

 

Table 2       Cell Number at each timepoint

 

Nominal loading concentration

(mg/L)

No.

Cell concentration (x104cells/mL)

0 h

24 h

48 h

72 h

Control

A

0.75

3.7

18

73

B

0.75

4.1

18

66

C

0.75

3.7

18

67

D

0.75

3.9

18

74

E

0.75

4.0

18

61

F

0.75

3.6

18

60

Mean

0.75

3.8

18

67

SD

0

0.20

0.24

5.9

3.1

A

0.75

4.0

17

58

B

0.75

4.0

18

51

C

0.75

3.5

17

49

Mean

0.75

3.8

17

52

SD

0

0.33

0.79

4.6

6.3

A

0.75

3.9

17

45

B

0.75

4.1

16

36

C

0.75

4.1

17

43

Mean

0.75

4.0

17

41

SD

0

0.14

0.24

4.9

13

A

0.75

3.9

17

54

B

0.75

3.8

19

70

C

0.75

3.8

18

62

Mean

0.75

3.9

18

62

SD

0

0.085

0.94

8.2

25

A

0.75

4.1

15

29

B

0.75

4.0

13

25

C

0.75

4.4

15

34

Mean

0.75

4.2

14

29

SD

0

0.21

0.95

4.2

50

A

0.75

3.9

8.3

17

B

0.75

3.9

8.8

17

C

0.75

3.9

9.2

19

Mean

0.75

3.9

8.8

18

SD

0

0.014

0.44

1.1

100

A

0.75

4.0

10

22

B

0.75

3.8

9.7

19

C

0.75

3.5

9.3

18

Mean

0.75

3.8

9.8

20

SD

0

0.26

0.49

2.1

 

Table 3       Growth rate and growth rate inhibition

 

Nominal loading concentration

(mg/L)

No.

Growth rate

(0-3 d)

Growth rate inhibition

(%)

Control

A

1.53

-

B

1.49

-

C

1.50

-

D

1.53

-

E

1.47

-

F

1.46

-

Mean

1.50

-

SD

0.0294

-

3.1

A

1.45

3.3

B

1.41

5.9

C

1.39

7.1

Mean

1.42

5.5*

SD

0.0289

1.9

6.3

A

1.37

8.7

B

1.29

14

C

1.35

9.8

Mean

1.34

11**

SD

0.0407

2.7

13

A

1.43

4.7

B

1.51

-1.2

C

1.47

1.6

Mean

1.47

1.7

SD

0.0442

3.0

25

A

1.21

19

B

1.18

21

C

1.27

15

Mean

1.22

19**

SD

0.0473

3.2

50

A

1.04

31

B

1.05

30

C

1.08

28

Mean

1.06

29**

SD

0.0198

1.3

100

A

1.13

25

B

1.07

28

C

1.07

29

Mean

1.09

27**

SD

0.0339

2.3

* significant difference vs control (p<0.05)

** significant difference vs control (p<0.01)

 

Table 4 Measured concentrations of calcium in test item solutions

 

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

Control a

4.75

4.91

4.89

4.61

Control b

4.69

4.90

4.81

4.67

3.1

4.68

-

-

4.61

6.3

4.73

-

-

4.52

13

4.67

-

-

3.97

25

4.72

-

-

3.97

50

4.81

-

-

4.21

100

4.90

5.11

4.98

4.26

n.d.: 1.26 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

 

Table 5 Measured concentrations of manganese in test solutions

 

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

Control a

0.112

0.110

0.106

0.107

Control b

0.117

0.110

0.107

0.106

3.1

0.116

-

-

0.101

6.3

0.116

-

-

0.102

13

0.109

-

-

0.0850

25

0.0990

-

-

0.0940

50

0.0797

-

-

0.0655

100

0.0415

0.0408

0.0403

0.0351

n.d.: <0.0314 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

Table 6 Measured concentrations of titanium in test solutions

 

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

Control a

n.d.

n.d.

n.d.

n.d.

Control b

n.d.

n.d.

n.d.

n.d.

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

n.d.

n.d.

n.d.

n.d.

n.d.: <0.0313 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

Table 7 Measured concentrations of calcium calculated by subtracting the calcium concentration (average) of control

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

0.187

0.205

0.134

n.d.

n.d.: <0.123 mg/L*5

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

*5Calcium concentration of the control was analysed 5 times, and the standard deviation of the control calcium concentration was calculated. Three times of the standard deviation was regarded as the determination limit of calcium concentrations derived test item after subtracting control calcium concentration.

 

Table 8 Measured concentrations of manganese calculated by subtracting the manganese concentration (average) of control

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

n.d.

n.d.

n.d.

n.d.

n.d.: <0.00268 mg/L*6

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

*5Manganese concentration of the control was analysed 5 times, and the standard deviation of the control manganese concentration was calculated. Three times of the standard deviation was regarded as the determination limit of manganese concentrations derived test item after subtracting control manganese concentration.

 

Table 9 Measured concentrations of titanium in test solutions

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

n.d.

n.d.

n.d.

n.d.

n.d.: <0.00313 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

Table 10 CaO concentrations calculated from the calcium concentrations

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

0.262

0.286

0.187

n.d.

 

n.d.: <0.172 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

Table 11 MnO2concentrations calculated from the manganese concentrations

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

0.262

0.286

0.187

n.d.

 

n.d.: <0.00424 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

Table 12 TiO2concentrations calculated from the titanium concentrations

Nominal loading concentration (mg/L)

Measured concentration (mg/L)

At the start

24 hours

48 hours

At the end

3.1

n.d.

-

-

n.d.

6.3

n.d.

-

-

n.d.

13

n.d.

-

-

n.d.

25

n.d.

-

-

n.d.

50

n.d.

-

-

n.d.

100

0.262

0.286

0.187

n.d.

 

n.d.: <0.00522 mg/L

:- no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

Table 13 Test item concentrations calculated from CaO, MnO2and TiO2concentrations

Nominal loading concentration (mg/L)

Measured concentration (mg/L)*7

(Percentage of measured concentration versus that at start

At the start

24 hours

48 hours

At the end

Geometric mean

3.1

n.d.

-

-

n.d.

0.006259

6.3

n.d.

-

-

n.d.

0.01259

13

n.d.

-

-

n.d.

0.02509

25

n.d.

-

-

n.d.

0.05009

50

n.d.

-

-

n.d.

0.1009

100

0.262

0.286

(109)

0.187

(71.5)

n.d.*8

0.200

n.d.: <0.172 mg/L (CaO), 0.00424 mg/L (MnO2), 0.00522 mg/L (TiO2)

-: no measurement because measured concentrations of the test item was below the determination limit at the start of exposure

 

*7Total of CaO, MnO2and TiO2. It was expressed as n.d. when all measured concentrations were n.d.

*8In reference to OECD Guidance Document No. 23, half the determination limit of the CaO (0.172 mg/L) was adopted for the calculation of geometric mean, because the CaO was detected but not quantified

*9Reference values calculated from the geometric rate and the measured concentration of the 100 mg/L exposure level

Validity criteria fulfilled:
yes
Conclusions:
The 72 h ErLC50 and ErC50 to green algae, under the conditions of this test was >100 and >0.2 mg/L, respectively. Statistically significant reduced growth rates were observed in all treatment groups, with the excpetion of 13 mg/L (nominal), compared to the control. The 72h NOEL and NOEC for growth rate was therefore established as 13 and 0.025 mg/L, respectively.
Executive summary:

OECD 201 (2018) - In a 72 hour toxicity study,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal loading rates of 3.1, 6.3, 13, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 22 ± 1 °C. The test item solutions were prepared by stirring weighed amounts of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter to produce water accommodated fractions. Analysis of the test preparations at 0 hours showed measured test concentrations to range from <limit of detection (LOD) (in the 3.1-50 mg/L groups) to 0.262 mg/L (in the 100 mg/L group). At 72 h the test item could not be detected in any of the test solution groups. The chemical analysis was based on measured concentrations of calcium, manganese and titanium (i.e. components of the test item) using ICP-AES and internal calibration curves. A geometric mean measured concentration was established for the top concentration group using measured values taken at 0 h (0.262 mg/L), 24 h (0.286 mg/L), 48 h (0.187 mg/L) and 72 h (half the LOD = 0.086 mg/L). The geometric mean measured concentration was determined to be 0.200 mg/L. Mean measured concentrations of the other test groups were calculated from the 0.200 mg/L value, based on the geometric spacing factor of the concentrations. In summary, the geometric mean measured concentrations were established as 0.00625, 0.0125, 0.0250, 0.0500, 0.100 and 0.200 mg/L.

 

The initial cell density added to each treatment flasks was 0.75x104cells/mL at 0 h. Cell biomasses were determined, using a particle counter, at 24, 48 and 72 h.

 

The ErL50and ErC50were estimated to be greater than the highest tested concentration i.e. >100 and > 0.2 mg/L, respectively, since 50 % of inhibition of growth rate was not observed within the exposure concentrations. Statistically significant reductions in growth rate were observed in all treatment groups compared to the control group, with the exception of the 13 mg/L (nominal loading rate) group. Based on this information the 72 h NOEL and NOEC for growth rate were established as 13 and 0.0250 mg/L, respectively. 

 

Routine microscopical examination of the algae cells concluded that algae cells in the top two concentration groups appeared to be slightly “bloated”.

 

Substance was not detectable after 72 hours due to low solubility of the test item and degradation/transformation/flocculation in the test system. This was deemed acceptable and unavoidable.  The presence of EDTA may have also impacted chemical availability to the organism.Overall this toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.

Description of key information

Endpoints based on geometric mean measured values

72 h ErC50: >0.2 mg/L; OECD 201; Inoue, H. (2019)

72 h NOEC: 0.025 mg/L; OECD 201; Inoue, H. (2019)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.025 mg/L

Additional information

OECD 201 (2018) - In a 72 hour toxicity study, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal loading rates of 3.1, 6.3, 13, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 22 ± 1 °C. The test item solutions were prepared by stirring weighed amounts of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter to produce water accommodated fractions. Analysis of the test preparations at 0 hours showed measured test concentrations to range from <limit of detection (LOD) (in the 3.1-50 mg/L groups) to 0.262 mg/L (in the 100 mg/L group). At 72 h the test item could not be detected in any of the test solution groups. The chemical analysis was based on measured concentrations of calcium, manganese and titanium (i.e. components of the test item) using ICP-AES and internal calibration curves. A geometric mean measured concentration was established for the top concentration group using measured values taken at 0 h (0.262 mg/L), 24 h (0.286 mg/L), 48 h (0.187 mg/L) and 72 h (half the LOD = 0.086 mg/L). The geometric mean measured concentration was determined to be 0.200 mg/L. Mean measured concentrations of the other test groups were calculated from the 0.200 mg/L value, based on the geometric spacing factor of the concentrations. In summary, the geometric mean measured concentrations were established as 0.00625, 0.0125, 0.0250, 0.0500, 0.100 and 0.200 mg/L.

The initial cell density added to each treatment flasks was 0.75x104cells/mL at 0 h. Cell biomasses were determined, using a particle counter, at 24, 48 and 72 h.

 

The ErC50was estimated to be greater than the highest tested concentration > 0.2 mg/L since 50 % of inhibition of growth rate was not observed within the exposure concentrations. Statistically significant reductions in growth rate were observed in all treatment groups compared to the control group, with the exception of the 13 mg/L (nominal loading rate) group. Based on this information the 72 h NOEC for growth rate was established as 0.0250 mg/L.

 

Routine microscopical examination of the algae cells concluded that algae cells in the top two concentration groups appeared to be slightly “bloated”.

The substance was not detectable after 72 hours due to low solubility of the test item and degradation/transformation/flocculation in the test system. This was deemed acceptable and unavoidable.  The presence of EDTA may have also impacted chemical availability to the organism. This toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.