Registration Dossier

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:
The range-finding test was conducted between 27 and 30 July 2015 and the definitive test between 25 and 28 August 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:
(2006; Annex 5 corrected 28 July 2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(amended 2009)
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:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Floral Super
- Appearance: clear liquid
- Storage condition of test material: At room temperature flushed with nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples (2 mL, in duplicate) were taken from the control and each exposure level at the start of the test and after 24, 48 and 72 hours. In addition, the undiluted WSF was sampled at the start of the test

At the end of the exposure period, samples were taken from one replicate of each test concentration. For the highest test concentration a second replicate was analysed as well because the algal cell density was observed to be higher than in the other two replicates.

Samples were stored in the freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WSF
Floral Super was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test substance.
* test medium: adjusted M2
* loading rate: 100 mg/L
* stirring period: one day of magnetic stirring in a closed vessel to reach the maximum solubility of the test substance in the test medium
* phase separation: after stirring, the aqueous mixture was left to stabilize for approximately 70 minutes whereafter the clear and colourless Water Soluble Fraction (WSF) was siphoned out and used as the highest test concentration (range-finding test)
* test concentration range: lower test concentrations for both the range-finding test and the final test were prepared by subsequent dilutions of the WSF in test medium

Test organisms

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

ACCLIMATION
- Stock culture medium: M1
- Pre-culture: 3 to 4 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.

After preparation, volumes of 120 mL test solution were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

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:
Temperature was maintained between 22 and 23°C throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.3 - 7.8.
Nominal and measured concentrations:
Range-finding test:
Nominal test concentrations: 1.0, 10 and 100% of WSF prepared at 100 mg/L.
Measured test concentrations (at test initiation): 0.35 and 18 mg/L at 1.0 and 100% of the WSF, respectively

Final test:
Based on the results of the range-finding test the following nominal test concentrations were assigned to the final test: 0.32, 1.0, 3.2, 10 and 32% of WSF prepared at 100 mg/L.
Measured test concentrations (at test initiation, t=0): 0.11, 0.34, 0.99, 3.2 and 9.8 mg/L at 0.32, 1.0, 3.2, 10 and 32% of the WSF, respectively; 29.8 mg/L at 100% of WSF; 1.04 mg/L in a 3.2% solution of WSF without algae
Measured test concentrations (at end of test, t=72): < LOD in all test solutions except the 3.2% solution of WSF without algae, in which the analysed concentration was 0.32 mg/L

The effect concentrations are based on average measured exposure concentrations.


Details on test conditions:
TEST SYSTEM
- Test vessel:
120 mL glass conical flasks were used, each containing 120 mL of test preparation for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 2 (at 3.2% of WSF)
- 1 extra replicate of each test concentration and the control for sampling purposes

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

- Pre-culture and test medium: adjusted M2 (standard M2 medium was adjusted with a larger amount of NaHCO3 (300 mg/L), the addition of HEPES buffer (6 mmol/L) and a lower pH). The medium was prepared using reverse osmosis purified deionised water (Milli-RO, Millipore) and the pH adjusted to 7.1 ± 0.3.

- Illumination: continuously using TLD-lamps with a light intensity within the range of 77 to 80 µE.m-2.s-1 while constantly shaking.

- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities were determined. 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 cuvettes (path length =10 mm. Algal medium was used as blank.

- Results used to determine the conditions for the definitive study:
The results of the range-finding test showed that growth was reduced by 49% in tests solutions at 10% of WSF prepared at 100 mg/L. Based on this information test concentrations of 0.32, 1.0, 3.2, 10 and 32% of WSF were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 1.3-2.4
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 0.17-0.65
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: growth rate: 1.2 mg/L (95% CI: 1.1-1.2 mg/L)
- Other: >10% growth rate inhibition at and above 0.56 mg/L.

The 72h-ErC50 for the algal culture tested falls within the historical range at the test facility (0.82-2.3 mg/L).
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).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

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. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Measured test substance concentrations

At the start of the test, the measured test concentrations were 0.11, 0.34, 0.99, 3.2 and 9.8 mg/l at 0.32, 1.0, 3.2, 10 and 32% of the WSF, respectively. The lowest test concentration of 0.11 mg/L decreased below the limit of detection (LOD=0.06 mg/l) within 24 hours and all higher concentrations decreased to 26-71% of initial. The next following concentration of 0.34 mg/L decreased below the LOD within 48 hours and all higher concentrations decreased to 2-39% of initial. After 72 hours of exposure all test concentrations had decreased below the LOD. The measured concentration in the samples taken from the 3.2 mg/L concentration without algae also decreased but remained higher (31% of initial after 72 hours of exposure) then in the vessel with algae. As test concentrations were not maintained within 80 -120% of initial, average exposure concentrations were calculated (see Table 2).

Table 1: Concentrations of the test item in test medium - final test

Time of sampling
[hours]

Percentage of WSF1
[%]

Analysed concentration2
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

<LOD

 

 

0.32

 0.114

 

 

1.0

0.335

 

 

3.2

0.986

 

 

 3.23

1.04

 

 

10

3.22

 

 

32

9.76

 

 

100

29.8

 

 

 

 

 

24

0

<LOD

 

 

0.32

<LOD

n.a.

 

1.0

 0.0884

26

 

3.2

0.607

62

 

 3.23

0.799

77

 

10

2.29

71

 

32

6.94

71

 

 

 

 

48

0

<LOD

 

 

0.32

<LOD

n.a.

 

1.0

<LOD

n.a.

 

3.2

 <LOD

1.7

 

 3.23

0.707

68

 

10

0.799

25

 

32

3.76

39

 

 

 

 

72

0

<LOD

 

 

0.32

<LOD

n.a.

 

1.0

<LOD

n.a.

 

3.2

 <LOD

n.a.

 

 3.23

0.321

31

 

10

<LOD

n.a.

 

32

<LOD

n.a.

 

32

 <LOD

n.a.

 

 

 

 

1          Percentage of a water soluble fraction (WSF) prepared at a loading rate of 100 mg/L.

2          Samples were stored in the freezer (≤ -15°C) until the day of analysis.

3          Without algae.

4          Estimated value, calculated by extrapolation of the calibration curve.

LOD   The limit of detection (LOD) of the method was determined to be 0.06 mg/L taking a dilution factor of two into account.

n.a.     Not applicable.

Table 2: Measured concentrations versus nominal concentrations

Floral Super, % of WSF prepared at 100 mg/L

Measured concentration (mg/L)

Average conc. (mg/L)

t=0h

t=24h

t=48h

t=72 h

0.32

0.11

0.032

0.032

0.032

0.041

1.0

0.335

0.088

0.032

0.032

0.072

3.2

0.986

0.607

0.032

0.032

0.13

3.21

1.04

0.799

0.707

0.321

0.66

10

3.22

2.29

0.799

0.032

0.65

 32

 9.76

 6.94

 3.76

 0.032

1.7

1003

29.8

-

-

-

-

1 Without algae

2 A factor of 2 below the LOD

3 Undiluted WSF. Not used as a test concentration

- Not measured/not calculated

Inhibition results

Table 3: Percentage inhibition of growth rate (total test period) during the final test

 

Average conc. Floral Super

(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.610

0.0397

6

0.041

1.614

0.0280

3

-0.3

0.072

1.590

0.0517

3

1.2

0.13

1.567

0.0475

3

2.6

0.65

1.325

0.0553

3

17.7*

1.7

0.782

0.3095

3

51.4*

* Effect was statistically significant

Table 4: Percentage inhibition of yield during the final test

Average conc. Floral Super

(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

124.8

15.54

6

0.041

125.9

10.41

3

-0.9

0.072

117.7

18.32

3

5.7

0.13

109.8

15.39

3

12.0

0.65

52.8

9.28

3

57.7*

1.7

13.1

13.66

3

89.5*

* Effect was statistically significant

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%
Conclusions:
The ErC50, ErC10 and NOEC were 1.6, 0.45 and 0.13 mg/L.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: N1. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% of the water soluble fraction (WSF) prepared at a loading rate of 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 22 ± 1 °C. The test material solutions were prepared by magnetic stirring for 24 hours in a closed vessel. After a stabilization period of approximately, the clear and colourless WSF was siphoned out of the preparation vessel.

Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated HPLC-UV method. Measured concentrations decreased steadily throughout the course of the test: the lowest two concentrations were not detectable anymore after 48 hours and at test end none of the test concentrations were detected, except in the replicate without algae. Therefore average exposure concentrations were calculated and used for expression of endpoints. Nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% of the WSF prepaed at 100 mg/L corresponded with 0.041, 0.072, 0.13, 0.65 and 1.7 mg/L, respectively.

Statistically significant inhibition of growth rate and yield was found at and above the average exposure concentration of 0.65 mg/L. The ErC10 and ErC50 based on average exposure concentrations were 0.45 and 1.6 mg/L, respectively. The NOEC was 0.13 mg/L.