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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:
24 Aug 2018 - 06 Nov 2018
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 aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
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 the WAF prepared at a loading rate of 10 mg solids/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 individually prepared loading rates of 1.0 to 100 mg solids/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for three hours. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure, except for the highest WAF which was slightly hazy.
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 was not completely soluble in test medium at the concentrations tested. A correction factor of 2.6 was used to correct for the water content of the test item. Concentrations are expressed based on solids.
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 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: 7.1 - 7.7
At t=72 h: 8.0 - 8.2
Nominal and measured concentrations:
Nominal: WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32, 100 mg solids/L
Measured concentrations at the start of the test: 0.67 µg solids/L, 0.27, 25 and 139 mg solids/L in WAFs prepared at loading rates of 1.0, 10, 32 and 100 mg solids/L, respectively. See 'Details on results' for more information about the measured concentrations.
Based on the obtained results, the Time Weighted Average exposure concentrations were determined to be 0.015, 0.0025, 0.27, 210 and 21000 µg solids /L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg solids/L, respectively.
See 'Any other information on materials and methods' for calculation of TWA concentrations, and 'Any other information on results' for a table with the measured concentrations and TWA.
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: 185 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 all test concentrations 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 93 to 96 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Algal cell density was quantified by counting cells using a microscope and a counting chamber because undissolved material was observed to develop during the exposure period at WAF loading rates of 56 mg solids/L and above interfering with the spectrophotometric measurement of samples.

COMBINED LIMIT/RANGE FINDING STUDY
- Combined Limit/Range finding study test concentrations: nominal 1.0, 10, 100 mg solids/L
- Results used to determine the conditions for the definitive study: Yes, ErC50 is estimated to be between 10 and 100 mg solids/L
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed Jul 2018)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 6.1 - 8.5 mg solids/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
44 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 36 - 52 µg solids/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 147 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 1006 - 1309 µg solids/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.015 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Dissolved test substance corrected for water content (specified in report as "solids")
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 the four lowest test concentrations tested when compared to the control. At the highest test concentration, no algal cells could be observed.
- Undissolved material was observed to develop during the exposure period at WAF loading rates of 56 mg solids/L.
- Effect parameters were also calculated based on loading rates, see 'Any other information on results' for the table.
- Measured concentrations: The measured concentrations at the start of the test were 0.67 µg solids/L and 0.27, 25 and 139 mg solids/L in WAFs prepared at loading rates of 1.0, 10, 32 and 100 mg solids/L, respectively. The concentration measured in the WAF prepared at a loading rate of 3.2 mg solids/L was at the level of the carry-over within the analytical system. At the three lowest test concentrations, the measured concentrations were at the carry-over level of the analytical system after 24 and 72 hours of exposure. The concentrations measured in the two highest test concentrations were at 0.059-17% of the initial concentrations after 24 hours of exposure, and at 0.021-0.30% of the initial concentrations at the end of the test, respectively. The concentrations measured in the samples taken from solutions with algae were comparable to the concentrations measured in the samples without algae indicating that the presence of biomass had no influence on the test item concentrations.
Because the recoveries observed for the QC samples prepared during the analysis of the samples of the final test were inconsistent with the recoveries of the QC samples prepared for the preceding combined limit/range-finding test, it was decided to analyse the reserve samples to confirm the analytical results. The concentrations detected in the reserve samples were comparable to the concentrations detected in the first set of samples, with the QC samples following a similar pattern as observed during the validation of the analytical method as well as during the analysis of the first set of samples taken during the final test, i.e. that the recoveries of the test item increase with increasing test item concentration, potentially leading to an overestimation of the measured concentrations at higher concentration levels.
Based on the obtained results, the Time Weighted Average exposure concentrations were calculated using the results of the initial analysis of the samples taken during the final test. Where concentrations were detected at the level of the carry-over, it could not be ascertained whether this concentration originated from the test sample or the analytical system. Consequently, these concentrations were estimated as half of the limit of detection following a worst-case assumption.
- An additional test was performed to assess whether the disappearance of the test item could be attributed to adhesion of the test item to the test vessles. The results showed that in pre-incubated test vessels, the test item disappeared in a similar way as in the main test. Therefore, the disappearance of test item cannot be explained by biodegradation or adhesion of the test item to the test vessels. Scientifically it is not justified to use the NOEC based on the TWA concentration since it is not known whether the observed effects are due to the low measured concentrations of test substance. Therefore, the effect parameters for growth rate inhibition based on nominal loading rate is more relevant. The NOELR is 10 mg solids/L, the EL10 is 7.3 mg solids/L and the EL50 is 44 mg/L.
- 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 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 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.
ELx calculation: weibit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding loading rate
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Determination of Time Weighted Average Concentrations

Loading rate (mg solids/L)

Measured concentration (mg solids/L)

TWA conc.

(mg solids/L)

t=0h

t=24h

t=72 h

1.0

0.00067

0.0000025 #

0.0000025 #

0.000015

3.2

0.0000025 #

0.0000025 #

0.0000025 #

0.0000025

10

0.266

0.0000025 #

0.0000025 #

0.00027

10 *

0.298

0.0000025 #

0.0000025 #

0.00029

32

24.8

0.0147

0.00532

0.21

100

139

24.2

0.418

21

* without algae; # Concentration estimated as half of the LOD (LOD/2 = 0.0025 µg solids/L).

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


Loading rate (mg solids/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.738

0.0285

6

 

1.0

1.692

0.0682

3

2.6

3.2

1.649

0.0320

3

5.1#

10

1.705

0.0226

3

1.9#

32

1.560

0.0512

3

10*

100

0.000

0.0000

3

100*

* Effect was statistically significant.; #Effect was statistically significant but biologically not relevant (<10%).

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

Loading rate (mg solids/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.454

 

2.049

 

1.713

 

1.0

3

0.794

45

2.455

-20

1.828

-6.7

3.2

3

0.441

70

2.686

-31

1.821

-6.3

10

3

0.000

100

3.206

-56

1.909

-11

32

3

0.000

100

2.670

-30

2.009

-17

100

3

0.000

100

0.000

100

0.000

100


Table 4: Effect Parameters Based on Loading Rates

Parameter (mg solids/L)

NOEL*

NOEL#

EL10

EL20

EL50

Growth rate

Value

1.0

10

7.3

15

44

lower 95%-cl

 

 

6.1

13

40

upper 95%-cl

 

 

8.5

17

50

Yield

Value

<1.0

<1.0

8.4

15

35

lower 95%-cl

 

 

0.30

1.9

19

upper 95%-cl

 

 

17

25

62

cl: confidence limit;* based on statistical significance;#based on biological relevance.


Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The effect parameters for growth rate inhibition were based on nominal loading rate. The NOELR is 10 mg solids/L, the EL10 is 7.3 mg solids/L and the EL50 is 44 mg/L.
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, 10, 32, and 100 mg solids/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 0.015, 0.0025, 0.27, 210 and 21000 µg solids/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg solids/L. A concentration-related increase of growth rate inhibition was observed with increasing test concentration, resulting in 100% inhibition of growth rate at the highest test concentration. The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 1147 and 44 µg solids/L, respectively, based on Time Weighted Average exposure concentrations. The effect parameters for growth rate inhibition based on nominal loading rate were considered to be more relevant due to unexplained disappearance of the test item during the exposure period. The NOELR is 10 mg solids/L, the EL10 is 7.3 mg solids/L and the EL50 is 44 mg/L. 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, 10, 32, and 100 mg solids/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 0.015, 0.0025, 0.27, 210 and 21000 µg solids/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg solids/L. A concentration-related increase of growth rate inhibition was observed with increasing test concentration, resulting in 100% inhibition of growth rate at the highest test concentration.The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 1147 and 44 µg solids/L, respectively, based on Time Weighted Average exposure concentrations. The effect parameters for growth rate inhibition based on nominal loading rate were considered to be more relevant due to unexplained disappearance of the test item during the exposure period. The NOELR is 10 mg solids/L, the EL10 is 7.3 mg solids/L and the EL50 is 44 mg/L. The study met all validity criteria, and is considered reliable without restriction.

Key value for chemical safety assessment

EC50 for freshwater algae:
44 mg/L
EC10 or NOEC for freshwater algae:
7.3 mg/L

Additional information

In a 2018 study with the registered substance, the NOELR, EL10 and EL50 were 10, 7.3 and 44 mg solids/L, respectively, based on nominal loading rates. Since the EL10 value is lower than the NOELR, this value is used as worst-case key value for chemical safety assessment. The measured concentrations cannot be reliably used to express the effect parameters due to unexplained disappearance of the test item during the exposure period. This study confirms a 2008 study which found similar results based on nominal loading rates.