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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:
14 January 1997 - 31 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Frequency: At the start of the exposure (0 hr) and the end of the exposure (72 hr)
- Sampling Volume: 2.0 mL/ test vessel
Vehicle:
no
Details on test solutions:
Preparation of the test solution (1000 mg/L): A hundred mL of medium was put in the test vessel and after the additive amount of the stock solution had been removed, 2.0 mL of the stock solution was added.
Preparation of the stock solution (50000 mg/L): Five hundred mg of the test substance was weighed and dissolved in medium, then adjusted to 10 mL.
Medium was used for the control group.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC22662
- Source: Aseptically subcultured in testing laboratory, Originally from American Type Culture Collection on 20 June 1996

PRECULTURE
- Period: 3 days
- Culturing media and conditions: Same as test

OTHER
- Confirming the reproducibility of the test conditions:
- Reference substance: Potassium dichromate (Reagent grade)
- Results: 72-hr EbC50 = 0.41 mg/L (linear regression analysis)



Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Shake culture (100 rpm)
Hardness:
No data
Test temperature:
23 ± 2 °C (22.9 - 24.2 °C)
pH:
- At the start of the exposure: 7.9
- At the end of the exposure: 7.8
Nominal and measured concentrations:
- Nominal concentration: 1000 mg/L
- Measured concentrations: 1005 mg/L (101 % of Nominal) at the start of the exposure, 992 mg/L (99 % of Nominal) at 72 hours exposure
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flask with silicon rubber plug (aeratable)
- Material: Glass
- Size: 300 mL
- Fill volume: 100 mL
- Incubator: AGP-150RL (ITO SEISAKUSYO CO. LTD.)
- Equipment
- Optical microscope: FHT (Olympus Corporation)
- Cell counter: CDA-500 (TOA Medical Electronics Co., Ltd.)
- Electrolytic solution for cell couner: Cellpack (TOA Medical Electronics Co., Ltd.)
- pH Meter: Desktop pH meter / Ion meter 900 A (ORION)
- Thermometer: TNA-120 (Tasco Japan Co. Ltd.)
- illuminance meter: IM-2D (TOPCON CORPORATION)
- Initial cells density: 1 × 10^4 cells/mL
- Control end cells density: 1944600 cells/mL (increased 194 times)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, OECD medium (See also Table 1.)


OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity and quality: 4000 - 5000 lux (within variation of ± 20 %, Around water surface)

OBSERVATION AND MEASUREMENTS
- Determination of cell concentrations: The test solution (1.0 mL) was sampled at 24, 48 and 72 hr and mixed with electrolytic solution (9.0 mL), and the cell concentration was measured by cell counter.
- Test solution: At the preparation, pH was measured in a reserved vessel besides three replicates. At the end of the exposure, pH was measured in the one of test vessels. Temperature and illuminance were measured at least once a day.


RANGE FINDING STUDY
- Test concentrations: 300, 1000 mg/L
- No. of replicates: 1
- Results: Growth rates for 72 hours in 300 mg/L and 1000 mg/L groups were 102 % and 112 % of control group, respectively.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 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.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
CELL GROWTH
- Control group: Increased to 194 times of initial cell density during 72 hours exposure (showed normal growth under the test conditions).
- 1000 mg/L group: Increased to 202 times of initial cell density during 72 hours exposure.

There were no environmental factors which might have affected the reliability of the test.
Reported statistics and error estimates:
- The test was conducted at one concentration level (1000 mg/L), and the median effective concentration value (EC50) could not be calculated as the growth inhibition rate (IA or Im) in this level was less than 50 %.
- The No observed effect concentration values (NOEC) were determined by Student's t-test (a = 0.05, two-tails), subsequent to F test (a = 0.05) for homogeneity of variances.

The toxicological values were calculated by nominal concentration because the measured concentrations of the test substance at the start of the exposure were kept within ± 20 % of the nominal concentration.

Table 5. Cell Density of Selenastrum capricornutum

Nominal concentration
mg/L
Vessel No. Cell Density (cells/mL)
0 24 48 72
Control 1 10000 66800 311600 1490900
2 10000 62500 283100 2171900
3 10000 61500 305200 2170900
Average 10000 63600 300000 1944600
SD 0 2800 15000 392900
1000 1 10000 61900 350500 2226900
2 10000 62800 321600 1623900
3 10000 60300 309200 2219900
Average 10000 61700 327100 2023600
SD 0 1300 21200 346100

SD = Standard deviation

Table 6. Growth Inhibition of Selenastrum capricornutum

Nominal Conc.
(Measured Conc.) *1
(mg/L)
No. Area *2
A (0-72h)
Inhibition (%)*5
IA(0-72h)
Rate*3
µ (24-48h)
Inhibition (%)*5
Im (24-48h)
Rate*4
µ (24-72h)
Inhibition (%)*5
Im (24-72h)
Control 1 26372000 0.0642 0.0647 -
2 33757000 0.0629 0.0739 -
3 34252000 0.0667 0.0742 -
Average 31460000 -
0.0646 -
0.0709 -
SD 4414000 0.0019 0.0054
1000
(1005)
1 36020000 0.0722 0.0746
2 28112000 0.0681 0.0678
3 34907000 0.0681 0.0751
Average 33013000 -4.9
0.0695 -7.6
0.0725 -2.3
SD 4281000 0.0024 0.0041

*1: Values in parentheses are the measured concentration at 0-hour.

*2: Area under the growth curves (0-72h)

*3: Growth rates (24-48h)

*4: Growth rates (24-72h)

*5: Values are the inhibition rates (%) based on the control.

SD: Standard deviation

* Indicates a significant difference (a = 0.05) from the control

(There was no sign in this test)

** Indicates a significant difference (a = 0.01) from the control

(There was no sign in this test)

Table 7. Calculated EC50 and NOEC

Based on IA(0-72 h) value (Areas under growth curve)
EbC50 (0-72) (mg/L) 95-Percent Confidence Limits (mg/L) NOECb (0-72) (mg/L)
> 1000 -- 1000
Based on Im (24-48 h) value (Growth rates)
ErC50 (24-48) (mg/L) 95-Percent Confidence Limits (mg/L) NOECr (24-48) (mg/L)
> 1000 -- 1000
Based on Im (24-72 h) value (Growth rates)
ErC50 (24-72) (mg/L) 95-Percent Confidence Limits (mg/L) NOECr (24-72) (mg/L)
> 1000 -- 1000

The EC50 values and associated 95 % confidence limits could not be determined by least squares linear regression analysis because the test was conducted at one concentration level.

The NOEC values were determined by Student's t-test (a = 0.05, two-tails), subsequent to F test (a = 0.05) for homogeneity of variances.

Calculation of the Growth Inhibition Rates

1. Growth curves

Growth curves were drawn by plotting the mean cell densities against time.

2. Calculation of the growth inhibition rates

1) Inhibition rates calculated from the area under the growth curve (IA)

A = (N1-N0)/2 × t1 + (N1+N2-2N0)/2 × (t2-t1) + ... + (Nn-1+Nn-2N0)/2 × (tn-tn-1)

A: Area under the growth curve

N0: Nominal cell density at the start of the exposure (cells/mL)

N1: Measured cell density at the time t1 (cells/mL)

Nn: Measured cell density at the time tn (cells/mL)

t1: Time at the first measurement of the cell density

tn: Time at the n-th measurement of the cell density

Growth inhibition rates were calculated by following formula:

IA = (Ac-At)/Ac × 100

Ac: Area under the growth curve of the control group

At: Area under the growth curve of the test group

2) Inhibition rates from comparison of the growth rates (Im)

µ = (lnNn-lnN1)/(tn-t1)

µ: Mean growth rate

N1: Measured cell density at the time t1 (cells/mL)

Nn: Measured cell density at the time tn (cells/mL)

t1: Time at the first measurement of the cell density

tn: Time at the n-th measurement of the cell density

Growth inhibition rates were calculated by following formula:

Im = (µct)/µc × 100

µc: Mean growth rates of the control group

µt: Mean growth rates of the test group

Validity criteria fulfilled:
yes
Remarks:
: Cell density at control group increased to 194 times of initial cell density during 72 hours exposure (showed normal growth under the test conditions).
Conclusions:
The 72-hr EbC50 was greater than 1000 mg/L(biomass) and the 72-hr NOECb was 1000 mg/L(biomass) for Pseudokirchnerella subcapitata.
Both the (24-72)-hr ErC50 was greater than 1000 mg/L(growth rate) and the (24-72)-hr NOECr was 1000 mg/L(growth rate) for Pseudokirchnerella subcapitata.
Executive summary:

Toxicity of diacetone alcohol was studied in one algae test. A study wih fresh water algae, Pseudokirchnerella subcapitata, was performed in a static system according to OECD TG 201 in compliance with GLP. A limit test was performed at 1000 mg/L. pH at the start of exposure was 7.9, pH at the end of exposure was 7.8 and the temperature was23 ±2 °C. Based on biomass, the 72 -hr EC50 was greater than 1000 mg/L and the 72 -hr NOEC was 1000 mg/L. Based on growth rate, the 72h-EC50 was greater than 1000 mg/L and the 72h-NOEC was 1000 mg/L. No toxic effect of diacetone alcohol was seen up to 1000 mg/L subtance.

Description of key information

An acute toxicity study was performed to assess the toxicity of DAA to freshwater algae species Pseudokirchnerella subcapitata (Mitsubishi Chemical Safety Institute Ltd., 1997). The study was conducted under GLP and according to OECD Test Guideline 201. One nominal concentration, 1000 mg/L, was tested as a the limit test. pH at the start of exposure was 7.9, pH at the end of exposure was 7.8 and the temperature was23 ±2 °C. Based on biomass, the 72 -hr EC50 was greater than 1000 mg/L and the 72 -hr NOEC was 1000 mg/L. Based on growth rate, the (24 -72)-hr EC50 was greater than 1000 mg/L and the (24 -72)-hr NOEC was 1000 mg/L. 

Three toxicity studies were conducted to assess the acute toxicity of DAA in Scenedesmus quadricauda and Microcystis aeruginosa (Bringmann and Kühn, 1977, 1978ab). The studies did not follow GLP and were not conducted according to any set guidelines. The studies were all rated ‘2’ for reliability under Klimisch standards. 

Bringmann and Kühn (1977, 1978a) evaluated the inhibition of cell proliferation in Scenedesmus quadricauda by DAA in a static freshwater system. Oxygen concentration was not measured. Growth was measured as relative turbidity at the beginning and at the end of the study. The authors reported that the toxicity threshold (8 d) of DAA in the S. quadricauda growth inhibition test is: 3000 mg/L in both study reports. 

Bringmann and Kühn (1978b) also evaluated the inhibition of cell proliferation in Microcystis aeruginosa by DAA in a static freshwater system. Growth inhibition was measured as relative turbidity at the end of the study (8 d) versus substance concentration. The authors reported that the toxicity threshold (8 d) of DAA in the M. aeruginosa growth inhibition test is: 530 mg/L. 

Key value for chemical safety assessment

Additional information