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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:
16 September 2013 - 19 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.
- Sampling method: Sampling was performed at the beginning of the experiment and thereafter 24-hour intervals.
- Sample storage conditions before analysis: The test item was extracted from the OECD media by Hexane, and then the extracts were immediately subjected to GC analysis.
- Others: Since the test item is evaporable, the sampling after 24 and 48 hour were done from separate replicate vessels for analytical sampling. At the end of the test, the analytical sampling and the observation was performed from the same vessel.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the fact, that the test item is poorly soluble in aqueous within the analytical method validation solution preparation method was assess to achieve the saturation concentration (study report 13/037-316AN). Based on these results, the test item was added into OECD media using 100 mg/L test item loading rate. This mixture was stirred vigorously for 4 hours at 20°C degree in tightly closed vessel in order to minimize losses due to evaporation. After 4 hours of equilibration, the mixture was centrifuged at 3000 rpm for 10 minutes, the mixture was poured into a separatory funnel and the undissolved test item was separated from the saturated solution after 15 minutes. The non-dissolved test material was removed by filtration through a fine (0.22 μm) to give the 100 % v/v saturated solution. Test solutions of lower test concentrations were prepared by appropriate dilution of this stock solution. During the analytical method validation the saturation concentration was found to be: 39.2 mg/L (for a nominal loading rate of 100 mg/L).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.0 – 23.3 °C
pH:
7.68 – 8.34
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.
Measured concentrations (geometric means): 1.73; 3.37; 6.72; 13.79 and 27.96 mg/L
Details on test conditions:
TEST SYSTEM
Taking into account that the test item is volatile the study was performed in a closed system followed the OECD recommendations. A simple filled closed bottle test with low algal densities was performed to minimize the loss of test substance from solution.
- Test vessel: Bottle
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: Absence of headspace.
- Initial cells density: 3.3x10+033 algal cells per mL
- Control end cells density:
- No. of vessels per concentration (replicates): 5 (3 for observations and 2 for analytical measurements)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, with bicarbonate enrichment.
- Detailed composition if non-standard medium was used:
The link between algal growth, the dissolved CO2 concentration and the pH of the algal medium is crucial for reproducible algal growth inhibition tests. To prevent such limitation of algal growth in a sealed test system, the study was performed based on the test procedure set forth in Mayer et al., 2000: The algae medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2), the pH was adjusted to 7.0 by addition of HCl, and the resulting CO2 concentration was sufficient to support algal growth in the absence of headspace.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes (see growth medium for details).
- Photoperiod: Continuously illuminated.
- Light intensity and quality: 8223 lux (equivalent to 111 μE/m2/s), ensured with fluorescent lamps (spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study (1): Open vessels, 2 replicates per dose, 3 replicates per control, 72 hours of exposure.
- Test concentrations: 0, 6.25, 12.5, 25, 50 and 100 % v/v saturated concentration
- Results used to determine the conditions for the definitive study: Inhibition of growth rate (%): 0, 0.2, 0.5, 1.0, 2.8 respectively.
- Range finding study (2): Closed vessels, 2 replicates per dose, 3 replicates per control, 72 hours of exposure.
- Test concentrations: 0, 6.25, 12.5, 25, 50 and 100 % v/v saturated concentration
- Results used to determine the conditions for the definitive study: Inhibition of growth rate (%): 0, 0.6, 0.8, 0.8 and 78.5 respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10.27 – 13.30
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
5.36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 4.59 – 6.26
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.93 – 4.35
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.37 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
- Unusual cell shape: There was no observed morphological deviations during the experiment.
Results with reference substance (positive control):
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 13/223-022AL) with the reference item Potassium dichromate is (Batch Number: 0769128): 21 - 24 August 2013.
The 72h ErC 50 : 0.88 mg/L, (95 % confidence limits: 0.80 – 0.97 mg/L)
The 72h EbC 50 : 0.55 mg/L, (95 % confidence limits: 0.51 – 0.61 mg/L)
The 72h EyC 50 : 0.49 mg/L, (95 % confidence limits: 0.45 – 0.54 mg/L)
Reported statistics and error estimates:
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period. The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test. The ErC50, EbC50 and EyC50 values of the test item were determined using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Results:

Nominal LR

(% V/V)

Measured

(mg/L)

0-72 h

Growth rate (µ) and % inhibition of µ

Area under the Growth Curves (A) and Percentage Inhibition of A

Yield (Y) and % inhibition of Y

µ

%

A

%

Y

%

Control

0.0

0.0807

0.0

0.0775

0.0

0.0714+

0.0

6.25

1.73

0.0751

7.0

0.0672*

13.3

0.0690+

3.3

12.5

3.37

0.0751

7.0

0.0624*

19.5

0.0674

5.36

25

6.72

0.0558*

30.8

0.0404+*

48.0

0.0539+*

24.6

50

13.79

0.0558*

30.8

0.0279*

64.0

0.0237*

66.8

100

27.96

0.0462*

42.8

0.0231*

70.2

0.0154*

78.4

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

+ : at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Parameter (0-72h)

Growth rate (mg/L)

Yield (mg/L)

Biomass (mg/L)

EC50 (95% CL)

11.69 (10.27-13.30

4.41 (3.96-4.91)

3.98 (3.49-4.54)

EC10(95% CL)

3.57 (2.93-4.35

2.37 (2.06-2.74)

1.79 (1.46-2.19)

EC20 (95% CL)

5.36 (4.59-6.26)

1.71 (1.43-2.06)

1.17 (0.91-1.52)

NOEC

3.37

Not determined (<1.73)

Not determined (<1.73)

LOEC

6.72

1.73

1.73

Concentrations of the test solutions:

Nominal

% v/v

Measured test item, mg/L

Stability

%

0 hours

24 hours

48 hours

72 hours

Control

Not detected

-

-

Not detected

-

100

34.45

32.25

31.81

17.29

50

50

14.76

16.87

15.85

9.16

62

25

6.96

7.75

8.04

4.69

67

12.5

3.53

3.63

3.76

2.69

76

6.25

1.96

1.85

1.92

1.28

65

The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item.

The stability of the test item in the biological system varied between 50-76%. The test item degrades in aqueous media producing Fenchyl alcohol as degradation product. Based on the ratio of the peak area corresponding to the Fenchyl alcohol and the test item, the lower test item concentration at the end of the algae test is due to the degradation of the test item to Fenchyl alcohol.

The biological results are based on the measured geometric mean concentrations.

Validity criteria fulfilled:
yes
Remarks:
(cell density in control increased by a factor of 56.50 (>16%), mean coefficient variation for section-by-section specific growth rates in control was 17.68% (<35%), coefficient of variation of average specific growth rates in control was 0.36% (<7%)).
Conclusions:
The 72h-EC50 was determined to be 11.69 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 3.37 mg/L.
Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a closed system, according to EU Method C3 and OECD Guideline 201. Based on preliminary range-finding studies, algae were exposed to nominal concentration of 0 (control, 6 replicates), 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (5 replicates each, 3 per observations and 2 per analytical monitoring). The corresponding measured geometric mean test item concentrations were 0, 1.73, 3.37, 6.72, 13.79 and 27.96 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item. The stability of the test item in the biological system varied between 50-76% since the test item degrades in aqueous media producing fenchyl alcohol as degradation product (based on the ratio of the peak area corresponding to the fenchyl alcohol and the test item). The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting chamber. All the validity criteria were fulfilled. The 72h-EC50 was determined to be 11.69 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 3.37 mg/L.

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
The analogue substance dextro alpha fenchyl acetate undergoes rapid hydrolysis to acetic acid and dextro alpha fenchyl alcohol which shares the same functional groups with the substance isoborneol and also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.19 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
growth rate
Remarks on result:
other: read-across from an analogue for which 72h-EC50 = 11.69 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
4.19 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
growth rate
Remarks on result:
other: read-across from an analogue for which 72h-EC20 = 5.36 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.79 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
growth rate
Remarks on result:
other: read-across from an analogue for which 72h-EC10 = 3.57 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.63 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
growth rate
Remarks on result:
other: read-across from an analogue for which 72h_NOEC = 3.37 mg/L
Validity criteria fulfilled:
not applicable
Conclusions:
Based on the read-across approach from the analogue fenchyl acetate, the 72h-EC50 of isoborneol was determined to be 9.19 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 2.63 mg/L.
Executive summary:

The effect of analogue substance fenchyl acetate was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a closed system, according to EU Method C3 and OECD Guideline 201. Based on preliminary range-finding studies, algae were exposed to nominal concentration of 0 (control, 6 replicates), 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (5 replicates each, 3 per observations and 2 per analytical monitoring). The corresponding measured geometric mean analogue substance concentrations were 0, 1.73, 3.37, 6.72, 13.79 and 27.96 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the analogue. The stability of the analogue in the biological system varied between 50-76% since it degrades in aqueous media producing fenchyl alcohol as degradation product (based on the ratio of the peak area corresponding to the fenchyl alcohol and the analogue). The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting chamber. All the validity criteria were fulfilled. The 72h-EC50 was determined to be 11.69 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 3.37 mg/L. Based on these results, the read-across was applied and the 72h-EC50 of isoborneol was determined to be 9.19 mg/L and the 72h-NOEC was 2.63 mg/L.

Description of key information

Key study. Read-across approach. Test method EU Method C3, OECD 201. GLP study. Based on the read-across approach, the 72h-EC50 of isoborneol was determined to be 9.19 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 2.63 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.19 mg/L
EC10 or NOEC for freshwater algae:
2.63 mg/L

Additional information

Key study. Read-across approach. The effect of fenchyl acetate was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a closed system, according to EU Method C3 and OECD Guideline 201. Based on preliminary range-finding studies, algae were exposed to nominal concentration of 0 (control, 6 replicates), 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (corresponding measured geometric mean 0, 1.73, 3.37, 6.72, 13.79 and 27.96 mg/L). The 72h-EC50 was determined to be 11.69 mg/L in algae (based on measured concentration and growth rate) in a closed static system. The 72h-NOEC was 3.37 mg/L. Based on these results, the read-across was applied and the 72h-EC50 of isoborneol was determined to be 9.19 mg/L and the 72h-NOEC was 2.63 mg/L.