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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:
12 Oct 2018 - 03 Jan 2019
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:
2006, Annex 5 corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and the control.
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WAF prepared at a loading rate of 1.0 mg/L 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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A 2-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for approximately 1 day. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. Lower test concentration in the range finding test and in the final test were prepared by diluting the WAF prepared at a loading rate of 1.0 mg/L.
All test solutions were clear and colorless at the end of the preparation procedure, with the exception of the WAFs prepared at loading rates of 10 and 100 mg/L in the combined limit/range-finding test, which were increasingly hazy. All test solutions in the combined limit/range-finding test were checked for the Tyndall effect using a laser pen. No light scattering was observed.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Other relevant information: The test item is a UVCB substance which was not completely soluble in test medium at the loading rate initially prepared.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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 21-24°C.
- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium 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.
- Stock culture medium: M1, according to NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”)
- Pre-culture medium and test medium: M2, according to OECD 201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22 - 24°C
pH:
At t=0 h: 8.2 - 8.3
At t=72 h: 7.8 - 7.9
Nominal and measured concentrations:
Nominal: Solutions containing 3.2, 10 and 32% of a WAF prepared at a loading rate of 1.0 mg/L and WAFs prepared at loading rates of 1.0, 3.2 and 100 mg/L.
Measured: 0.018, 0.063, 0.16, 0.78, 2.1 and 3.2 mg/L at start of the test in solutions containing 3.2, 10 and 32% of the WAF prepared at a loading rate of 1.0 mg/L and in WAFs prepared at loading rate of 1.0, 3.2, and 10 mg/L, respectively. The measured concentrations were estimated to decrease to 0.1 – 8% of initially measured at the end of the test.
Based on these results, the Time Weighted Average exposure concentrations were calculated to be 0.0089, 0.024, 0.059, 0.22, 0.75 and 1.2 mg/L, respectively. See 'Any other information on materials and methods' for details about the calculation of average exposure concentrations, and 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
- Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 184 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium according to OECD 201, formulated using tap water purified by reverse osmosis
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the WAF prepared at a loading rate of 1.0 mg/L and the control to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 81 to 86 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- 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 immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: yes, with WAF loading rates of 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: no, Combined Limit/Range finding study was not valid (mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35%). Subsequently, another Range finding study was performed with the following nominal test concentrations: solutions containing 1.0 and 10% of a WAF prepared at a loading rate of 1.0 mg/L and to WAFs prepared at loading rates of 1.0 and 10 mg/L. ErC50 was estimated to lie between WAFs at loading rates of 1.0 and 10 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed Nov 2018)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.50 - 0.54 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.22 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.19 - 0.24 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.059 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on statistical significance
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.22 mg/L when compared to the control.
- Measured concentrations were 59 – 103% of initially measured after 24 hours of exposure, and decreased to 0.1 – 8% of initially measured at the end of the test (based on extrapolated values).
Reserve samples collected from the WAFs prepared at loading rates of 3.2 and 10 mg/L at t=72 h were analysed because a too high dilution factor was applied to the initially analysed samples, resulting in concentrations below the calibration range. Reserve samples were only diluted in a 1:1 (v:v) ratio with methanol and analysed. However the analysed concentrations were again below the calibration range. Since results in the two sets of samples were comparable, the final exposure concentrations were calculated as average exposure concentrations of the two measurements. Additionally, small responses were detected in the controls at all sampling times. This was probably caused by carry over since a similar response was observed in the analytical blank. Therefore, it is considered to have no influence on the results of the test.
Based on these results, the average exposure concentrations were calculated and used to express effect parameters. Responses recorded at the end of the test were close to the responses measured in blank samples and should therefore be considered an estimation.
The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50 for growth rate inhibition (72h-ErC50) was 1.05 mg/L with a 95% confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
ECx calculation: probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Final Test: Test Samples Analysis Results

Time of sampling
[hours]

Loading rate (a)

[mg/L]

Concentration
analyzed (b)
[mg/L]

Relative to
initial
[%]

0

0

0.00099 (c)

 

 

0.032 (d)

0.0177

 

 

0.10 (d)

0.0625

 

 

0.32 (d)

0.161

 

 

1.0

0.781

 

 

1.0 (e)

0.715

 

 

3.2

2.14

 

 

10

3.16

 

24

0

0.0013  (c)

n.a.

 

0.032 (d)

0.0164

92

 

0.10 (d)

0.0494

79

 

0.32 (d)

0.148

92

 

1.0

0.460

59

 

1.0 (e)

0.567

79

 

3.2

2.04

95

 

10

3.27

103

72

0

0.0011 (c)

n.a.

 

0.032 (d)

0.0015 (f)

8

 

0.10 (d)

0.0015 (f)

2

 

0.32 (d)

0.00089 (f)

0.6

 

1.0

0.0015 (f)

0.2

 

1.0 (e)

0.00062 (f)

0.09

 

3.2

0.0037 (f)

0.2

 

10

0.022 (f)

0.7

72 (g)

3.2

0.0018 (f)

0.1

 

10

0.0065 (f)

0.2

(a) A water accommodated fraction (WAF) prepared at the loading rate.

(b) In a chromatogram of a blank sample peak at test item position is observed therefore it was not possible to determine limit of detection.

(c) Estimated value, calculated by extrapolation of the calibration curve. This is probably caused by carry over since a similar response was observed in the analytical blank.

(d) Test samples were prepared by dilution of the WAF prepared at a loading rate of 1.0 mg/L.

(e) Without algae.

(f) Estimated value, calculated by extrapolation of the calibration curve.

(g) Reserve samples.

n.a. Not applicable.

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period


TWA conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.737

0.0249

6

 

0.0089

1.742

0.0203

3

-0.28

0.024

1.732

0.0355

3

0.30

0.059

1.784

0.0130

3

-2.7

0.22

1.578

0.0497

3

9.2#

0.75

0.465

0.0828

3

73*

1.2

0.258

0.0371

3

85*

* - effect was statistically significant,#- effect was statistically significant however biologically not relevant (<10%).

Table 3: Growth Rate and Percentage Inhibition at Different Time Intervals 

TWA conc. (mg/L) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.714

 

1.850

 

1.649

 

0.0089

3

1.817

-6.0

1.784

3.6

1.627

1.4

0.024

3

1.672

2.4

1.841

0.50

1.684

-2.1

0.059

3

1.757

-2.6

1.904

-2.9

1.691

-2.5

0.22

3

1.529

11

1.544

17

1.663

-0.82

0.75

3

0.492

71

0.269

85

0.636

61

1.2

3

0.186

89

0.006

100

0.583

65

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 72h-EC50 for growth rate inhibition (ErC50) was 0.52 mg/L, the ErC10 was 0.22 mg/L. The NOErC was 0.059 mg/L based on statistical significance and 0.22 mg/L based on biological relevance.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to individually prepared Water Accommodated Fractions of the test item prepared at nominal loading rates of 1.0, 3.2 and 10 mg/L and to solutions containing 32, 10 and 3.2% of the WAF prepared at a loading rate of 1.0 mg/L (3 replicates per concentration), and an untreated control (6 replicates). Measured concentrations were not stable throughout the exposure period, therefore Time Weighted Average concentrations were calculated to be 0.0089, 0.024, 0.059, 0.22, 0.75 and 1.2 mg/L. No significant differences in growth rate were recorded at the three lowest concentrations, while a statistically significant increase in inhibition of growth rate was observed with increasing concentrations at the three highest test concentrations.

The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 0.52 and 0.22 mg/L, respectively. The 72h-NOErC was 0.059 mg/L based on statistical significance and 0.22 mg/L based on biological relevance.

The study met all validity criteria, and is considered reliable without restriction.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to individually prepared Water Accommodated Fractions of the test item prepared at nominal loading rates of 1.0, 3.2 and 10 mg/L and to solutions containing 32, 10 and 3.2% of the WAF prepared at a loading rate of 1.0 mg/L (3 replicates per concentration), and an untreated control (6 replicates). Measured concentrations were not stable throughout the exposure period, therefore Time Weighted Average concentrations were calculated to be 0.0089, 0.024, 0.059, 0.22, 0.75 and 1.2 mg/L. No significant differences in growth rate were recorded at the three lowest concentrations, while a statistically significant increase in inhibition of growth rate was observed with increasing concentrations at the three highest test concentrations.

The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 0.52 and 0.22 mg/L, respectively. The 72h-NOErC was 0.059 mg/L based on statistical significance and 0.22 mg/L based on biological relevance.

The study met all validity criteria, and is considered reliable without restriction.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.52 mg/L
EC10 or NOEC for freshwater algae:
0.22 mg/L

Additional information