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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:
2017-10-17 to 2018-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 23 March 2006 (Annex 5 corrected 28 July 2011)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
01 March 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10.0, 3.70 1.37, 0.51, 0.19 mg/L.
- Sampling method: For determination of the test item concentrations samples were taken from each test concentration and from the control at the start of the test and from each test concentrations after 24 h, 48 h and at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions used in the test were prepared by mechanical dispersion. For preparation of the test solutions a stock solution (10 mg/L) was first prepared by dissolving an amount of 0.01 g test item into 1000 mL test medium (OECD medium). This stock solution was placed into an ultrasonic bath for 10 min. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution.
After the formulation procedure algal cells were immediately introduced into the test solutions.
Untreated control ran parallel in the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum) (Printz-Starr).
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany.
- Method of cultivation: The stock cultures are small algal cultures that are regularly planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2 - 4 days. (The pre-culture was incubated for four days at this test.)

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
22.4 – 23.2 °C
pH:
7.96 – 8.95
Dissolved oxygen:
no data
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 10.0, 3.70 1.37, 0.51, 0.19 mg/L.
Calculated geometric mean concentrations: 6.03, 2.10, 1.04, 0.15 and 0.06 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel (material, size, fill volume): Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Aeration: No
- Initial cells density: 10^4 cells/mL
- Control end cells density: 95.0 × 10^4 cells/mL (mean value) after 72 hours.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
The test was performed with 2 extra replicates per test concentrations for analytical determinations.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD guideline

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 24 hours illumination.
- Light intensity and quality: Average light intensity of 8026 lux, which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Other: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.7
- Range finding study: A non-GLP preliminary test was performed at the nominal concentrations of 100, 10, 1, 0.1, and 0.01 mg/L. Untreated control ran parallel in the test. Algal cells were exposed to each concentration of the test item plus control, for 72 hours. The test was performed with two replicates in treatment groups and three replicates in the control. For preparation of test solution 0.04 g test item was dissolved in 400 mL OECD medium using 10 min ultrasonic dispersion. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution. In the preliminary range-finding test 100% inhibition was determined in the two highest test concentrations.
- Results used to determine the conditions for the definitive study: Based on a non-GLP Preliminary Range-Finding Test (see above), five test concentrations in a geometric series (with a spacing factor of 2.7) were used in the main test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 0.814 – 1.028
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.07 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 0.976 – 1.158
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 1.237 – 1.455
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 0.426 – 0.761
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% cong. limits: 0.626 – 0.869
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 0.963 – 1.121
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No
- Shape of the Algal Cells: No morphological abnormalities were observed in the control and in the concentrations of 0.06 and 0.15 mg/L (measured) during the test. Some swollen and thin cells were observed together with the normal cells in the concentration of 1.04 mg/L (measured) at the 48-h and 72-h observation period. In the concentrations of 2.10 and 6.03 mg/L (measured) thin cells were observed and in the highest concentration (in two replicates) the shape of algal cells could not be evaluated at 72 hours due to the absence of cells.
Results with reference substance (positive control):
The 72h ErC50: 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L)
The 72h EyC50: 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)
Reported statistics and error estimates:
For the determination of the LOEC and NOEC, Dunnett T3 test (α = 0.05) was used and for the determination of the ECx values Probit analysis was used (by SPSS software).

Analytical Results

Five test concentrations in a geometric series (with a spacing factor of 2.7) as a dilution series of a stock solution were investigated in the main test and analysed by HPLC method with UV detection at the start, after 24 h, 48 h and and at the end of the test.

Exposure concentrations were calculated as the geometric mean of the measured concentrations.

Table 1: Calculation of exposure concentrations

Nominal concentrations (mg/L)

Measured concentration [mg/L]

Geometric mean [mg/L]

start

24 h

48 h

end

Control

not detected

-

-

-

0.191

0.13

0.08

0.08

0.01*

0.06

0.512

0.50

0.14

0.15

0.05**

0.15

1.37

1.20

1.22

0.91

0.88

1.04

3.70

3.16

2.68

1.81

1.28

2.10

10.0

9.23

6.51

6.68

3.30

6.03

Remark: LOQ= 0.50 mg/L.

1: Due to the analytical sampling preparation (see Table 1 Appendix 2) at this concentration level the LOQ corresponds to LOQ/20

2: Due to the analytical sampling preparation (see Table 1 Appendix 2) at this concentration level the LOQ corresponds to LOQ/5

* (LOQ/20)/2

** (LOQ/5)/2

The analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours.

Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item MIKP had significant toxic effects on the growth of Raphidocelis subcapitata.
The 72-h EC10, EC20, EC50 for growth rate were determined to be 0.94, 1.07 and 1.33 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 0.65, 0.78 and 1.04 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 1.04 mg/L. The 72-h NOEC for growth rate and yield was determined to be 0.15 mg/L. The results are based on measured geometric mean test item concentrations.
Executive summary:

In a growht inhibition test according to OCCD guideline 201 and EU method C.3, the effect of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the growth of a unicellular green algal species was evaluated. Exponentially growing cultures of Raphidocelis subcapitata were exposed to the nominal test item concentrations of 10.0, 3.70 1.37, 0.51, 0.19 mg/L under defined conditions. Corresponding calculated geometric mean measured concentrations were: 0.06, 0.15, 1.04, 2.10 and 6.03 mg/L. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. Biological results are based on the measured geometric mean test item concentrations as the analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours.

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 0.94, 1.07 and 1.33 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 0.65, 0.78 and 1.04 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 1.04 mg/L. The 72-h NOEC for growth rate and yield was determined to be 0.15 mg/L. All validity criteria were met. The results are based on measured geometric mean test item concentrations.

Description of key information

In an algal growth inhibition test with Raphidocelis subcapitata, the 72-h EC10, EC20, EC50 for growth rate for MIKP were determined to be 0.94, 1.07 and 1.33 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 0.65, 0.78 and 1.04 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 1.04 mg/L. The 72-h NOEC for growth rate and yield was determined to be 0.15 mg/L. The results are based on measured geometric mean test item concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.33 mg/L
EC10 or NOEC for freshwater algae:
0.94 mg/L

Additional information

In a growth inhibition test according to OCCD guideline 201 and EU method C.3, the effect of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the growth of a unicellular green algal species was evaluated. Exponentially growing cultures of Raphidocelis subcapitata were exposed to the nominal test item concentrations of 10.0, 3.70 1.37, 0.51, 0.19 mg/L under defined conditions. Corresponding calculated geometric mean measured concentrations were: 0.06, 0.15, 1.04, 2.10 and 6.03 mg/L. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. Biological results are based on the measured geometric mean test item concentrations as the analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours.

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 0.94, 1.07 and 1.33 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 0.65, 0.78 and 1.04 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 1.04 mg/L. The 72-h NOEC for growth rate and yield was determined to be 0.15 mg/L. All validity criteria were met. The results are based on measured geometric mean test item concentrations.