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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March - 23 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2009, amended)
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, December 14, 2000
Deviations:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material in report: IFF 09-0035
- Physical state: Clear liquid
- Storage condition of test material: At room temperature protected from light
- Storage condition of test material:

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t = 24 and t=72 h
Volume 3.0 mL
Storage Samples were stored in a freezer until analysis.

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 demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start and at the end of the test period.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of IFF 09-0035 tested was a clear liquid nd the substance was not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test substance.
Preparation of test solutions started with a loading rate of 100 mg/L applying 23 hours of magnetic stirring to ensure reaching maximum dissolution in test medium. The resulting emulsion was subsequently left to settle for approximately 2½ hours resulting in a clear and colourless solution with a floating layer of undissolved test item. The Water Soluble Fraction (WSF) was then collected by means of siphoning. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 mL (combined range-finding limit) or 120 mL (final test) were added to each replicate of the respective test concentration. Subsequently, 1.0 or 2.4 mL (comb. limit/range-finder and final test, respectively) of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Stock culture: 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.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm

ACCLIMATION
3 days before the start of the test, cells from the algal stock culture were inoculated in 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21.7 - 22.9 °C
pH:
The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.3 - 7.8
Dissolved oxygen:
Not determined
Salinity:
Not determined
Nominal and measured concentrations:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test:
Nominal test concentrations: WSFs prepared at 1.0, 3.2, 10, 32 and 100% of a 100 mg/L loading rate
TWA mean measured test concentrations: 0.33, 0.85, 3.1, 11 and 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
Definitive test:
120 mL, all-glass, all-glass, airtight closed and completely filled (no headspace) were used. The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
1 x 10^4 cells/mL

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

- Illumination:
Continuously using TLD-lamps with a light intensity within the range of 79 to 82 µE.m-2.s-1.

- Incubation:
Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

- Determination of cell concentrations:
Samples were taken at 0, 24, 48 and 72 hours. 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 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

- Results used to determine the conditions for the definitive study:
Only slight effects on growth rate and yield were observed during the combined limit/range-finding test with WSFs prepared at 1.0, 10 and 100% of a 100 mg/L loading rate.
Based on these results samples taken from the WSF prepared at 100 mg/L were analysed. The initial concentration was 54 mg/L. This concentration rapidly decreased below 0.1 mg/L within 24 hours of exposure.. The rapid decrease was thought to be related to volatility of the test substance. As a consequence it was decided to continue with a final test using closed test vessels without headspace. The range for the final test was set between 1.0 and 100% WSF.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
10 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Growth rates were in the range of the controls at the two lowest concentrations during the 72-hour test period, whereas the growth rate of algae exposed to 0.85 mg/L and higher were increasingly reduced.
Statistically significant inhibition of growth rate was found at concentrations of 0.85 mg/L and higher, however, the effect observed at 0.85 mg/L and 3.1 mg/L was considered biologically insignificant (<10%) and therefore the NOEC was set to 3.1 mg/L.

Inhibition of yield increased with increasing concentration of IFF 09-0035 from 0.85 mg/L upwards resulting in 100% inhibition at 32 mg/L. Statistically significant inhibition of yield was found at test concentrations of 0.85 mg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.1 to 1.5 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 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 or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Percentage inhibition of growth rate (total test period) during the final test

IFF 09-0035

TWA (mg/l)

Mean

Std. Dev.

n

%Inhibition

Control

1.673

0.0255

6

0.0

0.33

1.677

0.0131

3

-0.2

0.85

1.635

0.0248

3

2.3*#

3.1

1.597

0.0434

3

4.5*#

11

1.390

0.0025

3

16.9*

32

0.000

0.0000

3

100.0*

* - effect was statistically significant

#- effect biologically insignificant (<10%)

 

Table 2: Percentage inhibition of growth rate at different time intervals during the final test

IFF 09-0035

TWA (mg/l) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.754

0.0

1.832

0.0

1.434

0.0

0.33

3

1.576

10.1

1.909

-4.2

1.545

-7.8

0.85

3

1.665

5.1

1.888

-3.1

1.352

5.7

3.1

3

1.831

-4.4

1.595

13.0

1.366

4.7

11

3

1.460

16.8

1.394

23.9

1.316

8.2

32

3

0.895

49.0

-0.621

133.9

-0.274

119.1

At the start of the test, the actual test concentrations were 0.52, 1.6, 5.6, 18 and 60 mg/l in the test groups representing 1.0, 3.2, 10, 32 and 100% WSF, respectively. Despite using closed systems without headspace, measured concentrations decreased by approximately 30% during the first 24-hour period and by 70 to 80% during the entire 72-hour test period. Based on these results, the average exposure concentrations were calculated (see Table 3). The range tested based on TWA (Time Weighted Average) concentrations corresponded with 0.33, 0.85, 3.1, 11 and 32 mg/L. The measured concentrations were comparable between the replicates with and without algae in the test group representing 10% WSF, indicative that the decrease of test substance in the test solution during the test period was not caused by adsorption to algae. 

 

Table 3            Measured concentrations versus nominal concentrations

Test group

IFF 09-0035 (%WSF) 

Measured concentration (mg/l

TWA (mg/L)

t=0h

t=24h

t=72 h

1.0

0.523

0.402

0.177

0.33

3.2

1.58

1.12

0.337

0.85

10

5.62

3.77

1.38

3.1

32

17.5

12.7

5.52

11

 100

60.1

 40.5

 13.4

 32

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See Overall remarks
Conclusions:
The ErC50, ErC10 and NOEC based on TWA concentrations were 14, 10 and 3.1 mg/L, respectively
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata.The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, the algae were exposed to 1.0, 3.2, 10, 32 and 100% WSF solutions of the test material prepared at a loading rate 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 21 - 23 °C.

Preparation of test solutions started with a loading rate of 100 mg/L applying 23 hours of magnetic stirring to ensure reaching maximum dissolution in test medium. The resulting emulsion was subsequently left to settle for approximately 2½ hours resulting in a clear and colourless solution with a floating layer of undissolved test item. The Water Soluble Fraction (WSF) was then collected by means of siphoning. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 mL (combined range-finding limit) or 120 mL (final test) were added to each replicate of the respective test concentration. Subsequently, 1.0 or 2.4 mL (comb. limit/range-finder and final test respectively) of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Samples were taken from all treatments at t = 0 , 24 and 72h and analysed with a validated GC-FID method. At the start of the test, the actual test concentrations were 0.52, 1.6, 5.6, 18 and 60 mg/l in the test groups representing 1.0, 3.2, 10, 32 and 100% WSF, respectively. Despite using closed systems without headspace, measured concentrations decreased by approximately 30% during the first 24-hour period and by 70 to 80% during the entire 72-hour test period. Based on these results, the average exposure concentrations were calculated. The range tested based on TWA (Time Weighted Average) concentrations corresponded with 0.33, 0.85, 3.1, 11 and 32 mg/L.

The measured concentrations were comparable between the replicates with and without algae in the test group representing 10% WSF, indicating that the decrease of test substance in the test solution during the test period was not caused by adsorption to algae.

Statistically significant inhibition of growth rate was found at measured concentrations of 0.85 mg/L and higher, however, the effect observed at 0.85 and 3.1 mg/L was considered biologically insignificant (<10%) and therefore the NOEC based on TWA concentrations was set to 3.1 mg/L.

The ErC10 and ErC50 based on TWA concentrations were 10 and 14 mg/L, respectively.