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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken at the begin of the experiment and after 72 hours.
Vehicle:
yes
Details on test solutions:
A stock suspension of 100 mg test substance per L was prepared by suspending 50 mg test substance in 500 mL test water by ultrasonic treatment and intense stirring before the start of the test. The suspension was diluted with test water to a ratio 1:10. Adequate volumes of the solution were added to test water to prepare the test concentrations.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Senedesmus subspicatus CHODAT, strain 86.81 SAG, Sammlung von Algenkulturen, Pflanzenphysiologisches Institut, Unviversity of Göttingen, Germany. Algae had been grown in the test laboratory under standardised conditions according to test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 ± 0.2 °C
pH:
The pH of the test water was adjusted to pH 7.0 with HCl. However, at the start of the study the pH range of the test media was from 6.6 to 7.0
Nominal and measured concentrations:
Nominal test concentrations: 0.010, 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L.
Mean measured concentrations: nominal concentrations of 0.01, 0.032 and 0.1 mg/L not measured; 0.12, 0.47 and 2.2 mg/L.
Details on test conditions:
TEST SYSTEM
- Type: closed, vessels covered with glass dishes
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks containing 15 mL algal suspensions
- Initial cells density: 10000 cells/mL
- Control end cells density: 411000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 7700 to 8400 Lux, using fluorescent tubes (universal white L 25, 36 W)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate
- Determination of cell concentrations: samples were taken from all test flasks after 24, 48 and 72 hours. Algae cell densitities were determined with an electronic particle counter (AL CELLCOUNTER, Model 871, AL-Systeme, Karlsruhe, Germany).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate

Mean measured concentrations were 0.12, 0.47 and 2.2 mg/L in the nominal 0.32, 1.0 and 3.2 mg/L treatments. no measurements were amde on the lower concentrations as these were below the NOEC.

The results of the study are based on the mean measured concentrations.

Table 1 - Cell densities determined over the 96 hour exposure period.

Nominal conc. (mg/L

Cell density (cells/mL x 10000)

0 hour

24 hours

48 hours

72 hours

Blank

1

3.36

9.24

41.11

0.01

1

3.10

7.61

46.38

0.032

1

3.13

7.99

45.82

0.10

1

2.99

7.32

43.49

0.32 (0.12*)

1

2.93

9.91

40.67

1.0 (0.47*)

1

2.56

6.60

26.32

3.2  (2.2*)

1

2.89

6.87

24.24

* mean measured concentration

Mean area under the growth curve was 736 in the control. Mean areas under the growth curve in the 0.01, 0.032, 0.10, 0.12, 0.47 and 2.2 mg/L treatments were 754, 757, 709, 736, 476 and 465 which, compared to the control gave inhibition of -2.4, -2.9, 3.6, -0.1, 35.3 and 36.8%.

The 72 hour EbC50was calculated to be >2.2 mg/L the corresponding NOEC was 0.12 mg/L.

The 0 – 72 h growth rate (µ) was 1.23 in the control. The 0 – 72 h growth rate (µ) in the 0.01, 0.032, 0.10, 0.12, 0.47 and 2.2 mg/L treatments were 1.27, 1.27, 1.25, 1.22, 1.09 and 1.06 which, compared to the control gave inhibition of -2.8, -3.5, -1.6, 0.7, 11.6 and 13.7%.

The 72 hour ErC50was calculated to be >2.2 mg/L the corresponding NOEC was 0.12 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Based on the information reported an acceptable assessment of growth inhibition to algae was achieved.
Executive summary:

A growth inhibition test was conducted under GLP to OECD TG 201 with freshwater green algae (Scenedesmus subspicatus), which were exposed under static conditions for 72 hours to cloquintocet-mexyl. The results of the study are based on the mean measured exposure concentrations. The nominal test concentrations were 0.010, 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L. For the highest three test concentrations, the mean measured concentrations were determined to be 0.12, 0.47 and 2.2 mg/L. On the basis of the mean area under the growth curve the growth inhibition for the different test concentrations was -2.4, -2.9, 3.6, -0.1, 35.3 and 36.8%. The 72 hour EbC50 was calculated to be >2.2 mg/L, and the corresponding NOEbC was 0.12 mg/L. On the basis of the growth rate, the inhibition of growth in the treated algal cultures was -2.8, -3.5, -1.6, 0.7, 11.6 and 13.7%. The 72 hour ErC50 was calculated to be >2.2 mg/L, and the corresponding NOErC was 0.12 mg/L.

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis for were taken at the start of the experiment and after 72 hours.
Vehicle:
yes
Details on test solutions:
100 mg of test substance and 40 mg of alkylphenol-polyglykolether were mixed and made up to 1005.8 g with water. This suspension was diluted to 1.8 mg test substance/L with water. Calculated amounts of stock solution were given into test water and homogeneously distributed to produce the desired test concentrations.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Strain: Cambridge 276/20
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 2 °C
pH:
7.5 (at start) - 9.4 after 72 hours (vessel with blank control); 7.8 to 8.0 - 7.7 to 8.7 after 72 hours (vessels with test substance)
Nominal and measured concentrations:
Nominal: 0, 0.022, 0.067, 0.2, 0.6, 1.8 mg/L
Measured (at start): 0, 0.02, 0.056, 0.153, 0.526, 1.76 mg/L
Measured (after 72 hours): 0, <0.005, <0.005, 0.02, 0.135, 1.13 mg/L
Measured (mean): 0, 0.01, 0.03, 0.09, 0.33, 1.45 mg/L
Details on test conditions:
TEST SYSTEM
- Type: closed, stoppered with aluminium caps
- Material, size, headspace, fill volume: 100 mL Erlenmeyer flasks, 50 mL test solution
- Initial cells density: 1.16E04 cells/mL
- Control end cells density: 1.19E06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 120 µE/m2sec ± 20% (approximately 8000 Lux), cold white fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density, counted daily
- Determination of cell concentrations: TOA cell counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study
- Test concentrations: not reported
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: derived by the registrant
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: derived by the registrant
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.09 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: derived by the registrant
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.42 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: this value was reported in an amendment of the study report. It is considered wrong by the registrant.

The results of the study are based on the nominal test concentrations.

pH ranged between 7.8 and 8.0 at the start of the study and between 7.7 and 9.4 at the end of the exposure period.

Table 1 - Cell densities determined over the 96 hour exposure period.

Nominal conc. (mg/L

Cell density (cells/mL x 10000)

0 hour

24 hours

48 hours

72 hours

Blank

1.16

4.4

26.6

119

0.022

1.16

5.3

27.5

99

0.067

1.16

4.4

20.3

108

0.20

1.16

5.6

23.1

95

0.60

1.16

4.5

8.3

39

1.8

1.16

5.4

8.1

30

Mean area under the growth curve was 2102 in the blank. Mean areas under the growth curve in the 0.022, 0.067, 0.20, 0.60 and 1.8 mg/L treatments were 1906, 1819, 1759, 706 and 614 which, compared to the blank gave inhibition of 9, 13, 16, 66 and 71%.

The 72 hour EbC50was calculated to be 0.56 mg/L the corresponding NOEC was 0.022 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal concentrations, the 72-hour EbC50 and NOEbC values were 0.42 mg/L and 0.2 mg/L. The re-calculated 72-hour ErC50 and NOErC values were 1.1 mg/L and 0.2 mg/L, respectively.
Executive summary:

The toxic effects of the substance to freshwater green algae (Scenedesmus subspicatus) was tested to OECD TG 201 under GLP in a 72-hour static study. A stock solution was prepared by using an appropriate solvent (alkylphenol-polyglykolether), distributing the test substance homogeneously in water. The nominal test concentrations of 0.022, 0.067, 0.2, 0.6 and 1.8 mg test substance/L were prepared by diluting appropriate amounts of the stock solution in test medium. The test concentrations were confirmed analytically at the start of the experiment and after 72 hours, demonstrating a considerable dissipation of test material during the test (measured concentrations were between <8 and 63% of nominal concentrations at the end of the test). Algal cultures in test medium were kept under continuous illumination for 72 hours. Six replicates of the blank control (test medium plus maximum concentration of solvent of 0.72 mg/L) and three replicates per nominal concentration were used. The initial cell density at the begin of the experiment was 11600 cells/mL. Cell densities in all test vessels were counted every 24 hours, and the areas under the growth curves were determined based on the average cell densities. The inhibition of the algal growth was determined on the basis of biomass. The 72-hour EbC50 value was 0.53 mg/L, and the 72-hour EbC0 value was <0.022 mg/L.

Description of key information

Lowest 72-hour ErC50 >2.2 mg/L (mean measured), green algae (OECD TG 201, Hertl 1996)


Lowest 72-hour NOEC = 0.09 mgL (mean measured), green algae (OECD TG 201, Rufli 1988)


Lowest 120-hour EbC50 for metabolite = 1.9 mg/L (nominal), blue algae ( ASTM - Guideline E 1218-90)


Lowest 120-hour NOEC for metabolite = 1.3 mg/L (nominal), blue algae ( ASTM - Guideline E 1218-90)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.09 mg/L

Additional information

Cloquintocet-mexyl was tested with three freshwater algae species, including green algae (Scenedesmus subspicatus), blue algae (Microcystis aeruginosa) and diatoms (Navicula pelliculosa).
In the key study, green algae were exposed to nominal concentrations of 0.022, 0.067, 0.2, 0.6 and 1.8 mg cloquintocet-mexyl/L, in a 72-hour static test (Rufli, 1988). This GLP compliant study was conducted to OECD guideline 201 and is considered reliable without restrictions. The original report presented the results for effects based on the biomass, and the 72-hour EbC50 value was 0.53 mg/L, and the 72-hour EbC0 value was <0.022 mg/L. Although there was a significant decline in the measured concentrations at the start of the experiment and after 72 hours of exposure (measured concentrations were <80% of nominal concentrations), the effect levels were based on nominal concentrations in the original report. In an amendment to the original study report, the effect levels were re-calculated for effects on biomass (using the areas under the growth curve) and growth rate (Rufli 2002). The respective 72-hour EbC50 and NOEbC values were 0.56 mg/L and 0.2 mg/L, whereas the respective 72-hour ErC50 and NOErC values were 1.11 mg/L and 0.2 mg/L. Also these effect levels were all based on the nominal test concentrations. The 72-hour no-effect concentration of 0.2 mg/L is regarded as reliable. However, the re-calculated ErC50 value may not be fully correct, as the overall inhibition of the growth rate was only about 30% at the highest nominal test concentration of 1.8 mg/L. This means that the ErC50 should be greater than the maximum tested concentration. Furthermore, the effect levels determined in the study should be based on the mean measured concentration, because these were less than 80% of the nominal test concentrations. The measured concentrations for the nominal test concentration of 0.2 mg/L were 0.153 mg/L at the start and 0.02 mg/L at the end of the experiment, giving a mean measured concentration of 0.09 mg/L. It can be concluded that the 72-hour NOErC value in the study by Rufli (1988) was 0.09 mg/L.
In a second study the influence of cloquintocet-mexyl on the growth of green algae (Scenedesmus subspicatus) was investigated in a 72-hour static test (Hertl and Schreitmüller, 1996). This GLP compliant study was conducted to OECD guideline 201 and is considered to be reliable without restrictions. Due to the pH-dependent instability of the test substance, the pH of the test medium was adjusted to about pH 7 at the start of the experiment. Results were expressed as mean measured concentrations. The growth-rate based ErC50 and NOErC values were determined to be >2.2 mg/L and 0.12 mg/L, respectively. As the 72-hour ErC50 value of >2.2 mg/L of this study is considered to be more reliable than the one derived from the previous study by Rufli (1988), this value will be considered in the hazard assessment.
A third GLP-compliant study conducted to ASTM guideline E 1218-90 with freshwater green algae (Scenedesmus subspicatus) is available (Grade, 1993). This study is considered to be reliable without restrictions. Algae were exposed to nominal concentrations of 0.047, 0.078, 0.13, 0.22, 0.37 and 0.61 mg cloquintocet-mexyl/L, in a 96-h static test. Results were expressed as nominal concentrations. The 96-h EbC50 and NOEbC values were 0.63 mg/L and 0.22 mg/L, respectively. An ErC50 value was not reported, but using the data given in the report in can be determined that the 96-hour ErC50 was >0.61 mg/L (based on nominal concentrations) or >0.24 mg/L (based on mean measured concentration, which was 0.47 mg/L at the begin and 0.01 mg/L after 96 hours), since the highest growth rate inhibition values calculated were approximately 14% (96-h) and 28% (72-h) for the highest concentration tested.


A 96-h static test was conducted to ASTM guideline E 1218-90 with freshwater blue-green algae (Microcystis aeruginosa) (Grade, 1993). This study is considered to be reliable without restrictions. Six nominal concentrations ranging from 1.1 to 13 mg/L were applied. The corresponding measured end concentrations ranged from 0.05 to 10.1 mg/L. No effects were observed at the three lowest concentrations tested. The 96-hour EbC50 (biomass) and NOEC values, based on measured concentrations at the end of the study, were 2.5 mg/L and 0.6 mg/L, respectively.


Toxicity to freshwater diatoms (Navicula pelliculosa) was investigated in a GLP compliant study conducted to ASTM guideline E1218-90 by Grade (1993). This study is considered to be reliable without restrictions. Algae were exposed to nominal concentrations of 0.40, 0.60, 1.1, 1.8 and 3.0 mg/L, in a 96-h static test. The 4-day EbC50 and NOEbC values were 1.7 mg/L and 0.4 mg/L, respectively, based on nominal test concentrations. 


Additionally, the toxicity of the main environmental metabolite of cloquintocet-mexyl has been investigated in three studies with freshwater green algae (Grade 1993), green-blue algae (Grade 1993b) and diatoms (Grade 1993c). The lowest endpoint values were obtained for green-blue algae, with EC50 and NOEC values of 1.9 and 1.3 mg/L, respectively, indicating similar or slightly lower toxic effects of the metabolite to freshwater algae and diatoms.