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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:
from 04 NOV 2008 to 27 MAY 2009
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 (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae), and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. All samples were deep-frozen immediately after sampling and protected from light until analysis.
Vehicle:
no
Details on test solutions:
The test medium of the highest nominal concentration of 100 mg/l was prepared by dissolving 40.2 mg of the test item completely in 402 ml of test water using intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare test media of the lower test concentrations. The test media were prepared just before the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Source (laboratory, culture collection): The algal culture is obtained from a commercial culture collection. The algae are cultivated in RCC’s laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An inoculum culture was set up three to four days before the start of the test.
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: no
- 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
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
Temperature maintained at 23°C during all the test (measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks)
pH:
The pH was measured and recorded in each treatment at the start and at the end of the test.
The pH varied between 8.1 and 8.2 at the start and 8.3 and 9.0 at the end (pH variation in the controls < 1.5 pH unit).
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/l
The measured concentrations of the test item in the test media of the test concentrations of 10 to 100 mg/L were between 95 and 105% of the nominal values at the start and the end of the test. This shows the correct preparation of the test media and the stability of the test item over the test periodof 72 hours.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed. Each test flask were covered with a glass dish.
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks containing 15 ml of test solutions
- Aeration: no
- the test solutions were continuously stirred by magnetic stirrers

- Initial cells density: 10000 cells/mL
- Control end cells density: determined by fluorescence measurement: mean of the 6 replicates: 208.5.10exp3 relative fluorescence units
- No. of organisms per vessel: not applicable
- 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: yes
- Adjustment of pH: after the preparation of the test media, the pH values were in the required range and no adjustment of pH was necessary.
- Photoperiod: the test flasks were continuously illuminated by fluorescent tubes installed above the test flasks.
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7200 Lux (range: 6300 to 7800 Lux, measured at nine places in the experimental area).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The biomass was determined by fluorescence measurement (BIO-TEK Multi-detection microplate reader). The measurement were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the test concentration of nominal 32 mg/l (chosen because at the higher nominal concentration of 100 mg/l, the algal cell density was too low for a reliable examination). The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2; this enlarged spacing factor was chosen because according to the results of the range-finding test, the concentration-effect relationship was rather flat and thus a large concentration range had to be tested
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study:a reduction cell number of 6%, 13% and 55% was observed at nominal test item concentrations of 1.0, 10 and 100 mg/l after 72H.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
61 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 59-63 mg/l
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The measured concentrations of the test item in the test media of the test concentrations of 10 to 100 mg/L were between 95 and 105% of the nominal values at the start and the end of the test. This shows the correct preparation of the test media and the stability of the test item over the test period of 72 hours.
The biological results were related to the nominal concentrations of the test item.
The test item had a significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the concentration of 32 mg/L and at all higher test concentrations.
Thus, this concentration was determined to be the 72 hour LOEC.
The 72-hour NOEC was determined to be 10 mg/L, since up to and including this test concentration the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Exponential growth in the control (for algal test): yes, the biomass increased by a factor of 177 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, Table 4) during 72 hours was 13%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.2%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.
Results with reference substance (positive control):
The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the historical range of Harlan Laboratories (72-hour EC50 for the growth rate: 1.20 mg/L (RCC Study No. B83755), range of the 72-hour EC50 for the growth rate from 2000 to 2008: 0.71–1.74 mg/L).
Reported statistics and error estimates:
Dunnett’s tests, one-sided, a=0.05

The biological results can be summarized as follows (on the basis of nominal concentrations of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

61

18

95% confidence interval

59 – 63

17 – 19

EC20  (mg/L)

>100

30

95% confidence interval

-

29 – 31

EC50  (mg/L)

>100

80

95% confidence interval

-

79 - 82

NOEC (mg/L)

10

10

LOEC (mg/L)

32

32

 

95% CI:      95% confidence interval

Validity criteria fulfilled:
yes
Remarks:
see above for controls andpH variation in the controls did not exceed 1.5 (since = 0.7 unit)
Conclusions:
Gaiacol is not considered as harmful for algae.
Executive summary:

In a 72 hour acute toxicity study (Harlan, 2009), the cultures of Pseudokirchneriella subcapitata, (formerly Selenastrum capricornutum), Strain No. 61.81 SAG were exposed to gaiacol at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L under static conditions in accordance with the OECD 201 guideline. The growth rate NOEC and EC50values based on cell density were 10 and >100 mg/L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -5.4 to 9.4 at 72H.

 

No abnormalities were noted.

Gaiacol was considered as not harmful for algae.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae toxicity study.

Description of key information

The growth rate NOEC and EC50 values based on cell density were 10 and >100 mg/L, respectively (Harlan, 2009: conducted in GLP, following OECD 201). 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

One key study has been selected regarding toxicity to algae (Harlan, 2009: conducted in GLP, following OECD 201).

The summary of this study is the following:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, (formerly Selenastrum capricornutum), Strain No. 61.81 SAG were exposed to gaiacol at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L under static conditions in accordance with the OECD 201 guideline. The growth rate NOEC and EC50 values based on cell density were 10 and >100 mg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from -9.4 to 5.4% at 72H.

No abnormalities were noted.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae toxicity study.

The other data available were all quoted as invalid because of strong deviation to OECD guidelines (durations too short or too long, temperature too high, not agreed species or endpoint tested...), but all led to EC50 > 100 mg/l.

Conclusion: Gaiacol is considered as not harmful to aquatic algae.