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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:
30 January 2017 to 03 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sampling method: At t = 0, t = 24 and t = 72 h, 2.0 mL was sampled. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were analysed on the day of collection.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONS
The batch tested was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest concentration of 100 mg/L applying 17 to 32 minutes of magnetic stirring to accelerate dissolution of the test material in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The colour of the final test solutions ranged from light pink to pink with increasing test concentrations.
After preparation, volumes of 30 mL were added to each replicate of the respective test concentration. Subsequently, 0.6 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Cultures were maintained for 3 days prior to the test

ACCLIMATION
- Culturing media and conditions (same as test or not): Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 to 24 °C. Light intensity was 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Stock culture medium was M1. 3 days before the start of the test, cells from the algal stock culture were inoculated in pre-culture medium M2 at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed: Not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22 to 24 °C
pH:
8.0 to 8.2
Nominal and measured concentrations:
- Nominal concentrations: 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L
- Time weighted average concentrations: 0.0046, 0.014, 0.041, 0.14 and 0.42 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL
- Type: Capped
- Material, size, headspace, fill volume: Plastic cell culture vessels (Greiner), containing 30 mL of test solution
- Aeration: No, however algal cells were kept in suspension by continuous shaking
- Initial cell density: 1 x 10^4 cells per mL
- Control end cell density: 1.85 x 10^6
- No. of vessels per concentration (replicates): 3; 1 or 2 replicates of each test concentration without algae and 1 or 2 extra replicates of each test group for sampling after 24 hours.
- No. of vessels per control (replicates): 5 replicates (5 instead of 6 replicates of the control treatment were available for analysis because one of replicates fell from the shaking table and possibly part of the solution was lost; sufficient number of replicates was present to perform statistical analysis with sufficient power)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L,CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L and NaHCO3 50 mg/L.
- Ca/mg ratio: Hardness (Ca + Mg) 0.24 mmol/L (24 mg CaCO3/L)
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature was continuously monitored in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 100 to 103 μE.m^-2.s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates as a background for treated solutions.
- Other: At the end of the test microscopic observations were performed on the 0.10 mg/L concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.1
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. The expected EC50 for both growth rate inhibition and yield inhibition was between 0.10 and 1.0 mg/L.

DATA HANDLING
- Calibration curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Comparison of average growth rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (lnXj - lnXi) / (tj - ti) (day^-1)
where:
μi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = [(µC - µT) / µC] x 100
where:
%Ir = percent inhibition in average specific growth rate
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

-Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = [(YC - YT) / YC] x 100
where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.020 - 0.030
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.016 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.015 - 0.016
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.014 - 0.015
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.009 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.008 - 0.010
Details on results:
Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 2 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarised in Table 3.
Growth rate was inhibited by 1.4 and 9.3 % at the two lowest concentrations at the end of the test. At concentrations of 0.041 mg/L and higher, growth rate was inhibited by 100 %. Effects observed at all concentrations were statistically significant and therefore, the NOEC for growth reduction based on statistical significance could not be determined in this study. As the effects observed at the two lowest concentrations were biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.014 mg/L.
Inhibition of yield increased from 7.4 % at the lowest concentration tested to 100 % at 0.041 mg/L and higher. All effects were statistically significant and therefore, the NOEC for yield reduction could not be determined in this study. As the effect observed at the lowest concentration was biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.0046 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to TWA concentration of 0.014 mg/L when compared to the control.

ACCEPTABILITY OF THE TEST
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 201).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 22 %).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 0.3 %).
Results with reference substance (positive control):
The reference test was carried out to check the sensitivity of the test system as used by the testing facility. Algae were exposed for 72 hours to K2Cr2O7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4.
Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72 h ERC50) was 1.2 mg/L with a 95 % confidence interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72 h ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72 h EYC50) was 0.43 mg/L with a 95 % confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. The observed 72 h EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test, α = 0.05, one-sided, smaller) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm, α=0.05, one-sided, smaller).
Calculation of ECx values was based on Weibull (growth rate) and probit (yield) analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA exposure concentrations of the test material.
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Measured concentrations versus nominal concentrations

Nominal concentration (mg/L)

Measured concentration (mg/L)

t = 0h

t = 24 h

t = 72 h

TWA conc. (mg/L)

0.046

0.046

0.0022

0.0015

0.0046

0.10

0.10

0.0096

0.0033

0.014

0.22

0.23

0.044

0.0029

0.041

0.22*

0.23

0.044

0.0019

0.040

0.46

0.48

0.16

0.032

0.14

1.0

1.1

0.55

0.11

0.42

* without algae

Table 2: Percentage inhibition of growth rate (total test period) during the final test

TWA concentration (mg/L)

Group Mean Growth Rate

Std. Dev.

n

% Inhibition

Control

1.768

0.0057

5

-

0.0046

1.742

0.0141

3

1.4#

0.014

1.604

0.0295

3

9.3#

0.041

0.000

0.0000

3

100*

0.14

0.000

0.0000

3

100*

0.42

0.000

0.0000

3

100*

*Effect statistically significant

#Effect statistically significant but biologically insignificant (<10 %)

 

Table 3: Percentage inhibition of growth rate at different time intervals during the final test 

TWA concentration (mg/L)

n

0 to 24 h

24 to 48 h

48 to 72 h

Mean

% Inhibition

Mean

% Inhibition

Mean

% Inhibition

Control

5

1.336

0.0

1.939

0.0

2.027

0.0

0.0046

3

1.766

-32.2

1.556

19.8

1.904

6.1

0.014

3

1.554

-16.3

1.431

26.2

1.826

9.9

0.041

3

0.0

100

0.0

100

0.0

100

0.14

3

0.0

100

0.0

100

0.0

100

0.42

3

0.0

100

0.0

100

0.0

100

 

Table 4: Percentage inhibition of yield during the final test 

TWA concentration (mg/L)

Group Mean Yields

Std. Dev.

n

% Inhibition

Control

199.9

3.45

5

-

0.0046

185.1

7.78

3

7.4#

0.014

122.2

10.68

3

39*

0.041

0.0

0.00

3

100*

0.14

0.0

0.00

3

100*

0.42

0.0

0.00

3

100*

*Effect statistically significant

#Effect statistically significant but biologically insignificant (<10 %)

Table 5: Effect parameters

 

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

Lower 95%-CI

Upper 95%-CI

0.014#

<0.0046*

0.014

0.014

0.015

0.017

0.016

0.019

0.023

0.020

0.030

Yield

Value

Lower 95%-CI

Upper 95%-CI

0.0046#

<0.0046*

0.009

0.008

0.010

0.011

0.010

0.011

0.016

0.015

0.016

# based on bilogical relevance

* based on statistical significance

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test material reduced growth rate significantly at 0.0046 mg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 0.023 mg/L with a 95 % confidence interval ranging from 0.020 to 0.030 mg/L. The EC50 for yield inhibition (72 h-EYC50) was 0.016 mg/L with a 95 % confidence interval ranging from 0.015 to 0.016 mg/L.
Executive summary:

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations were analysed. Measured concentrations were at the level of nominal at the start of the test (101 to 107 %). Measured concentrations decreased to 1.3 to 11 % of initial at the end of the exposure period. Based on these results, the Time Weighted Average (TWA) exposure concentrations were 0.0046, 0.014, 0.041, 0.040, 0.14 and 0.42 mg/L in nominally 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively.

Growth rate was inhibited by 1.4 and 9.3 % at the two lowest concentrations at the end of the test. At concentrations of 0.041 mg/L and higher, growth rate was inhibited by 100 %. Effects observed at all concentrations were statistically significant and therefore, the NOEC for growth reduction based on statistical significance could not be determined in this study. As the effects observed at the two lowest concentrations were biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.014 mg/L. 

Inhibition of yield increased from 7.4 % at the lowest concentration tested to 100 % at 0.041 mg/L and higher. All effects were statistically significant and therefore, the NOEC for yield reduction could not be determined in this study. As the effect observed at the lowest concentration was biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.0046 mg/L. 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material reduced growth rate significantly at 0.0046 mg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 0.023 mg/L with a 95 % confidence interval ranging from 0.020 to 0.030 mg/L. The EC50 for yield inhibition (72 h-EYC50) was 0.016 mg/L with a 95 % confidence interval ranging from 0.015 to 0.016 mg/L.

Description of key information

In an algae test according to OECD guideline 201, the test material reduced growth rate significantly at 0.0046 mg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 0.023 mg/L with a 95 % confidence interval ranging from 0.020 to 0.030 mg/L. The EC50 for yield inhibition (72 h-EYC50) was 0.016 mg/L with a 95 % confidence interval ranging from 0.015 to 0.016 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.023 mg/L
EC10 or NOEC for freshwater algae:
0.014 mg/L

Additional information

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations were analysed. Measured concentrations were at the level of nominal concentrations at the start of the test (101 to 107 %). Measured concentrations decreased to 1.3 to 11 % of initial at the end of the exposure period. Based on these results, the Time Weighted Average (TWA) exposure concentrations were 0.0046, 0.014, 0.041, 0.040, 0.14 and 0.42 mg/L in nominally 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively.

Growth rate was inhibited by 1.4 and 9.3 % at the two lowest concentrations at the end of the test. At concentrations of 0.041 mg/L and higher, growth rate was inhibited by 100 %. Effects observed at all concentrations were statistically significant and therefore, the NOEC for growth reduction based on statistical significance could not be determined in this study. As the effects observed at the two lowest concentrations were biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.014 mg/L. 

Inhibition of yield increased from 7.4 % at the lowest concentration tested to 100 % at 0.041 mg/L and higher. All effects were statistically significant and therefore, the NOEC for yield reduction could not be determined in this study. As the effect observed at the lowest concentration was biologically not relevant (i.e. <10 %) the NOEC based on biological relevance was set to 0.0046 mg/L. 

The study met the acceptability criteria described by the study plan and was considered valid.

Under the conditions of this study, the test material reduced growth rate significantly at 0.0046 mg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 0.023 mg/L with a 95 % confidence interval ranging from 0.020 to 0.030 mg/L. The EC50 for yield inhibition (72 h-EYC50) was 0.016 mg/L with a 95 % confidence interval ranging from 0.015 to 0.016 mg/L.