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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:
19 - 22 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany (03.01.2017)
Analytical monitoring:
yes
Remarks:
HPLC-DAD
Details on sampling:
- Concentrations: Control, 0.0625, 0.125, 0.250, 0.500, and 1.00 mg/L (nominal)
- Sampling method: All concentration levels and the control were analytically verified by HPLC-DAD at the start (0 h) and at the end of exposure (72 h) with algae.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of analysis, if necessary. Prepared samples were stored at room temperature in the autosampler until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared by dilution of a 10 mg/L stock solution of the test item, which was prepared in dilution water and stirred for 15 min at room temperature (1100 rpm).
- Controls: Six replicates of dilution water without test item, exposed under the same conditions as the test concentrations.
- Evidence of undissolved material: The stock solutions and all dilution levels were visually clear throughout the exposure period.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green algae
- Age of inoculum (at test initiation): A 3 d old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar and maintained in 24 h light per day at 2590 - 5180 lux (35 - 70 µE*m^-2*s^-1). The nutrient medium Z according to Lüttge et al. (1994) is used as culture medium.

ACCLIMATION
Not necessary, as the preculture is prepared in dilution water (OECD 201 medium).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
22.8 °C
pH:
7.52 - 7.95 (0 h)
7.70 - 8.85 (72 h)
Nominal and measured concentrations:
Control, 0.0625, 0.125, 0.250, 0.50, and 1.00 mg/L (nominal)
< LOQ, 0.0448, 0.0595, 0.0831, 0.151, and 0.295 mg/L (geom. mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL sterile Erlenmeyer flasks with cotton wool plugs
- Fill volume: 100 mL
- Initial cell density: 6839 cells/mL
- Control end cells density: 2547865 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, nutrient medium Z, according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline (OECD 201 medium)
- Culture medium different from test medium: Algae were cultured in nutrient medium Z according to Lüttge et al. (1994). For this study, a preculture was prepared in dilution water 3 d prior to the test start. Dilution water was prepared according to the guideline (OECD 201 medium).
- Intervals of water quality measurement: The pH at the start of exposure was measured in one additional replicate of every concentration level. At the end of exposure, the pH-value was measured from the pooled samples of each test item concentration and the control after measurement of cell density. Room temperature was continuously monitored.

OTHER TEST CONDITIONS
- Photoperiod: 24 h light/d
- Light intensity and quality: 5503 lux (mean)

EFFECT PARAMETERS MEASURED:
- Cell concentrations (chlorophyll a fluorescence): Every 24 h, using a fluorometer (Microplate Reader Chameleon V, Hidex), with an excitation wavelength of 436 nm and an emission wavelength of 685 nm.
- Other: No auto-fluorescence of the test item was identified.
- Microscopic evaluation: At the start and end of exposure (0 and 72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Two preliminary non-GLP range-finding tests were conducted.
- Test concentrations: 1, 10, and 100 mg/L (1st range-finding test); 0.001, 0.01, 0.1, and 1.00 mg/L (2nd range-finding test)
- Results used to determine the conditions for the definitive study: Yes, 100% growth rate inhibition after 72 h at 1.00 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 0.0657 - 0.0764 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.114 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.109 - 0.120 mg/L
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities: No morphological abnormalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test media were clear throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, results of the last reference test dating 18 - 21 Apr 2017 produced results in the valid range.
- ErC50 (72 h): 0.559 mg/L, 95% confidence interval: 0.538 - 0.581 mg/L
Reported statistics and error estimates:
EC values with confidence intervals were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by statistically significant differences of growth rates and yield. The following statistical tests were applied with ToxRat Professional Version 3.2.1: Shapiro-Wilk´s test on normal distribution (significance level 0.01), Levene´s test on variance homogeneity (significance level 0.01), and Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (significance level 0.05).
EC CALCULATION - GROWTH RATE AFTER 72 HOURS
Equation: sigmoidal dose-response (variable slope)
y = bottom + (top - bottom)/(1 + 10^((logEC50-x)*HillSlope))
Transform: x = log(x)
FITTING RESULTS
BEST-FIT VALUES
Bottom: -0.4245; Top: 102.3; logEC50: -0.4903; HillSlope: 3.565; EC50: 0.3201; EC10: 0.1754
GOODNESS OF FIT
Degrees of Freedom: 11; R square: 0.9933; Absolute Sum of Squares: 169.3; Sy.x: 3.463

VALIDITY CRITERIA

The present study met the validity criteria set out by the guideline (Table 1).

 

Table 1: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

A 373-fold increase in biomass was recorded in the control (specific growth rate 1.97/d).

Yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

The mean coefficient of variation in the control was 15.6%.

Yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.26%.

Yes

 

ANALYTICAL RESULTS

The measured concentrations of the test item were between 88 and 103% at the start of the exposure (0 h) and between < LOQ and 10% at the end of exposure (72 h) (Table 2). Since the measured concentrations were not within ± 20% of the nominal concentrations, effect concentrations were based on the calculated geometric mean measured concentrations.

 

Table 2. Measured concentrations and percentage of the nominal concentration of the test item in fresh media ( 0 h) and old media (72 h)

sampling time

0 h

fresh medium

72 h

old medium

 

nominal concentration of the test item [mg/L]

 

meas. conc. [mg/L]

 

%

 

meas. conc. [mg/L]

 

%

Geometric mean measured concentration

[mg/L]1)

1.00

0.927

93

0.0937

9

0.295

0.500

0.440

88

0.0517*

10

0.151

0.250

0.221

88

< LOQ

0.0831

0.125

0.113

91

< LOQ

0.0595

0.0625

0.0641

103

< LOQ

0.0448

Control

< LOQ

< LOQ

 

Meas. conc.      = measured concentration of the test item

%                         = percent of the nominal concentration of the test item

LOQ                     = limit of quantification (0.0625 mg/L test item)

*                          = < LOQ, but > 70% LOQ

1)                         = measured concentrations < 70% LOQ were taken into account with half of the LOQ (= 0.0625 mg/L)

 

BIOLOGICAL RESULTS

After 72 h of exposure growth inhibition occurred (Table 3).

All effect values were based on the geometric mean measured test item concentrations. The EC50 (72 h) with 95% confidence interval was 0.114 mg/L (0.109 - 0.120 mg/L) for the ErC50 (72 ) and 0.0815 mg/L (0.0626 - 0.106 mg/L) for the EyC50 (72 h). The EC10 (72 h) with 95% confidence interval was 0.0708 mg/L (0.0657 - 0.0764 mg/L) for the ErC10 (72 h) and 0.0771 mg/L (0.0601 - 0.105 mg/L) for the EyC10 (72 h). The NOEC values for both inhibition of growth rate and yield after 72 h were 0.0595 mg/L.

Table 3. Evaluation of cell growth after 72 hours. Statistically significant differences in growth rates and yield compared to control values are marked (+), differences that are not significant are marked (-).

geometric mean measured test item concentration

measured initial test item concentration

 

replicate

growth rate

inhibition of growth rate

yield

inhibition of yield

[mg/L]

[mg/L]

 

[d-1]

[%]

[cells/mL]

[%]

0.295

0.927

1

2

3

n.a.

n.a.

n.a.

n.a.

-0.29

100

-3982

100

-0.26

100

-3731

100

mean

(+) n.a.

100

(+) n.a.

100

0.151

0.440

1

2

3

0.37

81

13705

99

0.63

68

38731

98

0.40

80

105555

99

mean

(+) 0.46

76

(+) 22664

99

0.0831

0.221

1

2

3

1.52

23

653464

74

1.54

22

691385

73

1.62

18

864048

66

mean

(+) 1.56

21

(+) 736299

71

0.0595

0.113

1

2

3

1.97

0

2506441

1

1.97

0

2486954

2

1.95

1

2379277

6

mean

(-) 1.96

1

(-) 2457557

3

0.0448

0.0641

1

2

3

1.95

1

2365642

7

1.96

1

2457050

3

1.95

1

2353060

7

mean

(-) 1.95

1

(+)2391917

6

Control

 

1

2

3

4

5

6

1.98

 

2589130

 

1.97

2505095

1.97

2525928

1.97

2495498

1.98

2583746

1.97

2546761

mean

1.97

 

2541026

 

n.a.       = data not determinable

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The experimentally determined effect values for the toxicity to aquatic algae are an ErC10 (72 h) of 0.0708 mg/L and an ErC50 (72 h) of 0.114 mg/L (geometric mean measured, OECD 201, P. subcapitata).

Description of key information

ErC10 (72 h) = 0.0708 mg/L (geom. mean measured, OECD 202, P. subcapitata)

ErC50 (72 h) = 0.114 mg/L (geom. mean measured, OECD 202, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.114 mg/L
EC10 or NOEC for freshwater algae:
0.071 mg/L

Additional information

There is one study, in which the toxicity of2-Hydroxycyclohepta-2,4,6-trienone (CAS 533-75-5) to aquatic unicellular alga was assessed according to OECD guideline 201 and GLP.

In a static test, Pseudokirchneriella subcapitata at an initial cell density of 6839 cells/mL was exposed to five test item concentrations in a geometric series with a dilution factor of 2 ranging from 0.0625 – 1.00 mg/L (nominal) for 72 h. The concentrations of the test item in the test solutions were analytically verified by HPLC-DAD at the start (0 h) and end of exposure (72 h).

The measured concentrations of test item at the start of exposure (0 h) ranged from 88 – 103% of the nominal values over all tested concentration levels. The measured concentrations in the aged media (72 h) ranged from < LOQ to 10% of the nominal values. Therefore, all effect values were based on the geometric mean measured concentrations. After 72 h, growth inhibition was observed but no morphological abnormalities were recorded. The obtained ErC10 (72 h) is 0.0708 mg/L and the ErC50 (72 h) is 0.114 mg/L (geom. mean measured).