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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:
25 Jun 2018 - 01 Aug 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 individual loading rates ranging between 1.0 and 100 mg solids/L. A one-hour period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle for 30 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. A floating layer was observed at the surface of the WAFs prepared at a loading rate of 100 mg solids/L.
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: Test item was not completely soluble in test medium at the loading rates initially prepared. A correction was made for the purity/composition of the test item. A correction factor of 2.554 was used. Concentration/doses are expressed as 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: 8.2-8.4
At t=72 h: 8.2-8.4
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32, 100 mg solids/L
Measured concentrations decreased during the exposure time. Therefore, Time Weighted Average concentrations were calculated: 0.23, 0.87, 2.6, 22, 81 mg solids/L. See 'Any other information on materials and methods' for TWA calculation 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: 262 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 10 mg solids/L to observe for any abnormal appearance of the algae compared to the control.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 87 to 89 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: 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:
EC10
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 1.1-2.5 mg solids/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 11-16 mg solids/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: Based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.87 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
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 2.6 mg solids/L when compared to the control.
- Measured concentrations: The initial measured concentrations were 0.90, 2.9, 8.1, 27 and 98 mg solids/L, respectively, in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg solids/L. At the end of the test, the three lowest measured test concentrations had decreased below the LOQ (i.e. 0.01 mg solids/L), while the two highest measured test concentrations were at the level of 54 82% of initially measured. Small responses were measured in the analytical blank and in the control samples. The concentrations measured in the control samples at the start of the test and after 24 hours of exposure were comparable to the responses measured in the analytical blank and were considered caused by carry over, hence not relevant. The concentration measured in the control sample at the end of the test was higher than the analytical blank. The reason for this response is unknown. However, since the biological effects in the final test were comparable to the biological effects observed in the combined limit/range-finding test at similar test item concentrations and validity criteria for the control were met, it was assumed that the eventual presence of the test item at the measured concentration during the last phase of the exposure had negligible effect on the algal growth.
- 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: Step-down Jonckheere-Terpstra 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: Time Weighted Average Versus Loading Rate

Loading rate (mg solids/L)

Measured concentration (mg solids/L)

TWA (mg solids/L)

t=0h

t=24h

t=72 h

Control

0.000078 #

0.000073 #

0.00554

0.00045

1.0

0.900

0.510

0.00025 #

0.23

3.2

2.88

2.26

0.00025 #

0.87

10

8.06

7.63

0.00013 #

2.6

10 *

8.52 *

7.60 *

0.00027 *,#

2.7 #

32

27.2

26.5

14.6

22

100

98.3

76.2

81.0

81

* without algae, # estimated by extrapolation of the calibration curve.

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


TWA conc.
(mgsolids/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.854

0.0454

6

 

0.23

1.808

0.0291

3

2.48

0.87

1.807

0.0285

3

2.54*

2.6

1.510

0.0283

3

19*

22

0.764

0.0651

3

59*

81

0.156

0.2699

3

92*

* - effect was statistically significant.

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

TWA conc.

(mgsolids/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.025

 

1.879

 

1.657

 

0.23

3

2.095

-3.4

1.707

9.2

1.623

2.0

0.87

3

2.047

-1.1

1.758

6.5

1.616

2.5

2.6

3

1.714

15

1.450

23

1.367

17

22

3

1.408

30

0.144

92

0.741

55

81

3

1.166

42

-0.940

150

0.241

85

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The ErC10 and ErC50 were 1.7 and 13 mg solids/L, respectively. NOErC was 0.23 mg solids/L based on statistical significance and 0.87 mg solids/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, 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 0.23, 0.87, 2.6, 22 and 81 mg 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 at all test concentrations. A statistically significant growth rate inhibition was observed at the four highest WAFs tested, but a biologically relevant growth rate inhibition was observed only in the three highest WAFs tested. The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 13 and 1.7 mg solids/L, respectively. The 72h-NOErC was 0.23 mg solids/L based on statistical significance and 0.87 mg solids/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.

Description of key information

Two studies with substance analogues were performed. The 72 -h ErC(L)50 values were 13 mg solids/L and 44 mg solids/L and the ErC(L)10 values were 1.7 and 7.3 mg solids/L for C8 -C18 and C12 Amphoacetates, respectively. The NOErC for C8 -C18 was 0.87 mg solids/L. The lowest ErC50 value is used as a worst-case read-across value for the registered substance. Since the NOErC is lower than the ErC10, that value is used as a worst-case read-across value.

Key value for chemical safety assessment

EC50 for freshwater algae:
13 mg/L
EC10 or NOEC for freshwater algae:
0.87 mg/L

Additional information

The ErC10 and ErC50 were 1.7 and 13 mg solids/L, respectively. NOErC was 0.87 mg solids/L. This result is read across to the registered substance, the rationale is attached in Section 13.