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:
25 Jul - 08 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, undiluted filtrate (loading rate 100 mg/L), 1:2, 1:4, and 1:8 dilution of filtrate (Day 0 and Day 3)
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start of the test. For the sampling at the end of the test, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 30 mL) was too small to perform the analyses. A volume of 100 mL per sample was necessary for analytical purposes.
Immediately after sampling, methanol (10 mL methanol per 100 mL sample volume) was added to each sample to stabilize the latter during the storage period. The concentrations of the test item were analyzed in one of the duplicate test media samples from the dilutions 1:8, 1:4, 1:2 and the undiluted filtrate from the start and the end of the test. The samples from the dilution 1:16 were not analyzed, since this concentration was below the NOEC determined in this test. From the control, one of the duplicate samples was analyzed per sampling time.
- Sample storage conditions before analysis: Immediately after sampling, methanol (10 mL per 100 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low water solubility of the test item, a suspension with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 200.1 mg of the test item in 2000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer for three hours at room temperature in the dark to dissolve a maximum amount of the test item in test water. The stirring time was based on the stirring pre-experiment, which showed that the maximum amount of dissolved test item was reached after this stirring time. After stirring, the suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material.
The undiluted filtrate was used as highest test concentration. It was further diluted with test water to prepare the test media with the lower test concentrations.
- Controls: yes, test medium control

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany)
- Source: in-house culture at IEC Laboratories
- Age of inoculum (at test initiation): An inoculum culture was set up 4 d before the start of the exposure. The algae were cultivated under test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
- Method of cultivation: Algae were cultivated under standard conditions, according to the test guidelines.

ACCLIMATION
No acclimation. The algae were cultivated under test conditions.
- Culturing media and conditions: same as test

Study design

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

Test conditions

Hardness:
0.15 mmol/L = 15 mg/L as CaCO3
Test temperature:
22 °C
pH:
7.4 - 7.6 (control)
7.4 - 7.7 (treatments)
Nominal and measured concentrations:
control, 1:16, 1:8, 1:4, 1:2 dilutions, and undiluted filtrate (nominal loading rate of 100 mg/L)
< LOQ, n.a., 0.052, 0.075, 0.10, and 0.42 mg/L (geometric means measured at 0 h; n.a. = not analyzed because < NOEC)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: covered with a glass lid
- Material, size, headspace, fill volume: Size: 75 mL; Fill volume: 30 mL
- Initial cells density: 5000 cells/mL (nominal)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile, purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was visually controlled and recorded daily during the exposure period.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: LED light, 65 - 69 µE * s-¹ * m-²
- Other: The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen, Switzerland) and were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorimeter (SpectraMax I3x, Molecular Devices Ltd, Berkshire, UK)
- Chlorophyll measurement: excitation: 440 nm; emission: 680 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes, a stirring pre-experiment (GLP) was performed to determine the solubility of the poorly solutble test item (water solubility < 100 mg/L) in test water and to determine the optimum time period to reach equilibration within a reasonable time period.
- Test concentrations: Three individual suspensions of the test item with a loading rate of 100 mg/L were stireed for 3, 24, and 96 h.
- Results used to determine the conditions for the definitive study: The analytically determined concentration of dissolved test item in the filtrates were 0.36 mg/L (3 h), 0.25 mg/L (24 h), and 0.26 mg/L (96 h). These results showed that the maximum concentration of dissolved test item in test water was reached after a stirring period of 3 h.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.15 - 0.20 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: not determinable
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.055 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: 95% CI: 0.042 - 0.067 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: 95% CI: 0.22 - 0.29 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50 (72 h): 1.0 mg/L potassium dichromate (Jul 2016)
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test.
Statistical analysis was performed using ToxRat Professional®.

Any other information on results incl. tables

ANALYTICAL RESULTS

Table 1. Analytical results. The mean measured concentrations of the test item were calculated as the geometric mean of the concentrations measured at the start and the end of the test (72 h).

Treatment / Dilution

 

Analytical measured concentration
of the test item
[mg/L]

 

Mean measured concentration*
(geometric mean)

 

 

0 hours

72 hours

[mg/L]

Dilution 1:16

n.a.

n.a.

n.a.

Dilution 1:8

0.36

<LOQ

0.052

Dilution 1:4

0.75

<LOQ

0.075

Dilution 1:2

1.4

<LOQ

0.10

Undiluted Filtrate°

2.2

0.082

0.42

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L

LOQ: Limit of quantification (LOQ = 0.015 mg test item/L)

*: If the measured concentration was below LOQ, half of the LOQ (½ LOQ = 0.0075 mg/L) was used to calculate the mean measured concentration

n.a.: Not analyzed since below NOEC determined in this test

During the test period of 72 hours the test item concentrations in the test media decreased below the Limit of Quantification of the analytical method (LOQ = 0.015 mg/L) and 4 % of initial measurement (Table 1).

BIOLOGICAL RESULTS

Table 2. Biomass of Algae.

Treatment / Dilution Mean measured concentration [mg/L] Rep. no. Biomass of algae* (24 hours) Biomass of algae* (48 hours) Biomass of algae* (72 hours)
Control --- 1 1.6 13.9 68.2
2 1.7 12.4 72
3 2.3 13.7 74.1
4 2.6 14.3 74
5 1.7 12.6 75.5
6 1.7 13.4 79.9
Mean 2 13.4 73.9
SD 0.4 0.7 3.9
 1:16 n.a. 1 1.6 13.6 75.5
2 2.4 13.8 78
3 1.8 13.9 66.8
Mean 1.9 13.8 73.5
SD 0.4 0.2 5.9
 1:8 0.052 1 2 12.4 71.1
2 2.4 13.3 69.5
3 2.2 14.5 71.5
Mean 2.2 13.4 70.7
SD 0.2 1.1 1.1
 1:4 0.075 1 2.5 11.6 59.7
2 1.9 10.8 67.3
3 1.5 12.2 55.4
Mean 2 11.5 60.8
SD 0.5 0.7 6
 1:2 0.1 1 2.5 11.4 56.5
2 1.9 10.2 49.2
3 1.6 12.5 62.9
Mean 2 11.4 56.2
SD 0.5 1.2 6.9
Undiluted 0.42 1 1.7 5.2 24.2
 Filtrate° 2 1.5 5.9 26.1
3 1.5 5.4 25.9
Mean 1.6 5.5 25.4
SD 0.1 0.3 1

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L. SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.57 x 104 relative fluorescence units.

n.a.: Not analyzed since below NOEC determined in this test

Table 3. Average Growth Rate (µ)

Treatment / Dilution Mean measured conc. [mg/L] Average growth rate µ (day-1) and Inhibition of µ (Ir)  
0-24 h   0-48 h   0-72 h  
 

µ

Ir[%]

µ

Ir[%]

µ

Ir[%]

Control

---

1.208

0

1.574

0

1.619

0

 1:16

n.a.

1.206

0.2

1.588

-0.9

1.617

0.2

01:08

0.052

1.338

-10.8

1.574

0

1.605

0.9

01:04

0.075

1.218

-0.8

1.500*

4.7

1.553*

4.1

01:02

0.1

1.231

-1.9

1.492*

5.2

1.526*

5.7

Undiluted Filtrate°

0.42

0.997

17.5

1.129*

28.3

1.263*

22

*: mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller, α = 0.05)

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

n.a.: Not analyzed since below NOEC determined in this test

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a valid, reliable and conclusive study according to OECD TG 201, the impact of the test item 10-HSA on the growth of the freshwater green algal species Pseudokirchneriella subcapitata after an exposure of 72 hours was determined. The NOEC, EC10 and EC 50 were mean measured 0.052, 0.18 and > 0.42 mg/L for growth rate and mean measured 0.052, 0.055 and 0.25 mg/L for yield.
Executive summary:

The impact of the test item 10-HSA on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EU) No 2016/266, C.3.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared by using ultrasonic treatment for 15 minutes and intensive stirring for 3 hours to reach a maximum concentration of dissolved test item in test water. The stirring time was based on a pre-experiment. After stirring, the suspension was filtered through a 0.45 µm membrane filter. The undiluted filtrate with a loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate were used as test media. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The analytical measurements from test media at the start of the test show the correct preparation of the test media, i.e. the theoretical spacing factor of two between the different test concentrations was met. During the test period of 72 hours the test item concentrations in the test media decreased below the Limit of Quantification of the analytical method (LOQ = 0.015 mg/L) and 4 % of initial measurement. The mean measured concentrationsof 10-HSA were calculated as the geometric mean of the concentrations measured at the start and the end of the test (72 hours).

The biological results were as follows:

Growth rate:

EC10 (0-72 h) [mg/L]: 0.18 (95% confidence interval 0.15 – 0.20)

EC20 (0-72 h) [mg/L]: 0.37 (95% confidence interval 0.34 – 0.42)

EC50 (0-72 h) [mg/L]: > 0.42 (95% confidence interval not determined)

NOEC [mg/L]: 0.052

LOEC [mg/L]: 0.075

Yield:

EC10 (0-72 h) [mg/L]: 0.055 (95% confidence interval 0.042 – 0.067)

EC20 (0-72 h) [mg/L]: 0.092 (95% confidence interval 0.077 – 0.11)

EC50 (0-72 h) [mg/L]: 0.25 (95% confidence interval 0.22 - 0.29)

NOEC [mg/L]: 0.052

LOEC [mg/L]: 0.075