Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-04 to 2010-01-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Treatments: 0 (Control), 100 mg/L filtered dispersion and 1:3.2, 1:10, 1:32 and 1:100 dilutions thereof

- Sampling method: Duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. Immediately after sampling, each sample was spiked with 90 mL MeOH.

Due to the difficult analytical detection, additional samples were prepared as follows:
At the start and the end of the test, a second set of duplicate samples (without algae) was taken of each treatment. For this, additional flasks containing the test medium without algae were incubated under the test conditions until the end of the test. These samples were not spiked with MeOH.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.

The concentrations of the test item SILRES® BS 1701 were determined in the duplicate spiked test medium samples from the dilution 1:3.2 and the undiluted filtrate from test start and test end. The samples from the less concentrated dilutions were not analyzed, since these dilutions were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 150.15 mg of the test item in 1500 mL of test water. The dispersion was supported by ultrasonic treatment for 15 minutes and intense stirring by a magnetic stirrer over 96 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.

After the stirring period, the dispersion was let to settle for 15 minutes and was then filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The negative pressure of the filtration unit was reduced as far as possible. The test water (containing the water dissolvable part of the test item) was taken from the middle of the water column to avoid any undissolved test item in the test water. This undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)

- Strain: 61.81 SAG

- Source (laboratory, culture collection): The test organism used for the study were supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.


ACCLIMATION
- Acclimation period: An inoculum culture was set up four days before the start of the exposure.

- Culturing media and conditions (same as test or not): The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3
Test temperature:
21 and 22 °C
pH:
8.1 and 8.2
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal treatments: 0 (Control), 100 mg/L filtered dispersion and 1:3.2, 1:10, 1:32 and 1:100 dilutions thereof.

At the start of the test, the analytically determined concentrations of the test item in the test media (dilution 1:3.2 and the undiluted filtrate) were 280 and 1200 μg/L, respectively. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, the measured concentrations were both lower than the biological limit of quantification of the test item (LOQbio=10.1 μg test item/L). The mean measured concentrations in these two treatments were 38 and 78 μg/L.

The results are expressed relative to nominal loading rates and measured concentrations.
Details on test conditions:
TEST SYSTEM

- Test vessel: 50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labelled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

- Initial cells density: The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).

- Control end cells density: Not reported

- Replication: The test design included three replicates per test concentration and six replicates of the control.


TEST CONDITIONS

Temperature: The test flasks were incubated in a temperature-controlled water bath at a temperature between 21 and 22 °C.


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the required concentrations.

- 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 at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: No

- Photoperiod: Continuous

- Light intensity and quality: The flasks were illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above them. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7600 Lux (range: 7130 to 8240 Lux, measured at nine places in the experimental area). The light intensity over the incubation area (measured at nine places in the experimental area) was within ±15% from the average light intensity as recommended by the guideline.


EFFECT PARAMETERS MEASURED:

- Other: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate. Effects on algal growth rate and biomass (yield) were determined from these measurements.


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate but based on hydrolysis half-life exposure is likely to be mainly to hydrolysis product.
Basis for effect:
other: growth rate and biomass (yield)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate but based on hydrolysis half-life exposure is likely to be mainly to hydrolysis product.
Basis for effect:
other: growth rate and biomass (yield)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass (yield)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass (yield)
Results with reference substance (positive control):
72-hour EC50 for growth rate: 0.75 mg/L, range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71-1.74 mg/L
Reported statistics and error estimates:
The LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests

Table 1. Test results - Average Growth Rates (μ)

 

Treatment / Dilution (mg/L)

Mean measured concentration (μg/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

---

1.45

0.0

1.64

0.0

1.54

0.0

1:100

---

1.42

2.5

1.64

0.2

1.54

-0.3

1:32

---

1.40

3.7

1.63

0.5

1.55

-1.0

1:10

---

1.35

6.9

1.62

1.5

1.54

0.1

1:3.2

38

1.44

1.2

1.65

-0.3

1.54

-0.1

Undiluted Filtrate

78

1.41

3.0

1.57

4.2

1.41*

8.1

*: mean value significantly lower than in the control (according to Dunnett’s-tests, one-sided, α = 0.05)

 

Table 2. Test results - Yield (Y)

 

Treatment / Dilution (mg/L)

Mean measured concentration (μg/L)

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

 

 

Y

Iy (%)

Y

Iy (%)

Y

Iy (%)

Control

---

8.5

0.0

66.6

0.0

260.8

0.0

1:100

---

8.1

4.6

66.2

0.7

263.5

-1.0

1:32

---

7.9

7.1

65.4

1.8

271.4

-4.1

1:10

---

7.5

12.3

63.3

4.9

259.5

0.5

1:3.2

38

8.3

2.5

67.2

-0.9

260.5

0.1

Undiluted Filtrate

78

8.0

5.8

58.0

12.9

178.1*

31.7

*: mean value significantly lower than in the control (according to Dunnett’s-tests, one-sided, α = 0.05)

Validity criteria fulfilled:
yes
Conclusions:
A 72-h NOELR of 32 mg/L and LOELR of 100 mg/L have been determined for the effects of the test substance on growth rate and biomass (yield) of Pseudokirchneriella subcapitata. Loading rates above 0.1 mg/L are above the water solubility of the substance (<0.1 mg/L). The concentration of test substance in the test medium corresponding to the NOELR was 0.28 mg/L based on mean measured concentrations over the period of the test. It is therefore likely that the test medium contained predominantly dissolved hydrolysis products of the substance but these were not quantified. It is also possible that the presence of undissolved material may have contributed to the effects observed on growth rate and biomass in the undiluted dispersion however there is no conclusive evidence for this.

Description of key information

72-hour EC50 >1.2 mg/l and NOEC 0.28 mg/l (initial measured concentrations), growth rate and biomass of Pseudokirchneriella subcapitata. Loading rates above 0.1 mg/l are above the limit of solubility of the substance (<0.1 mg/l) and it cannot be excluded that undissolved test material was present. The results are considered to be > limit of solubility.

Key value for chemical safety assessment

Additional information

A 72-hour EC50 value of >1.2 mg/l and a NOEC value of 0.28 mg/l based on initial measured concentrations, and a 72-hour NOELR of 32 mg/l and LOELR of 100 mg/l based on nominal concentrations, have been determined for the effects of the test substance on growth rate and biomass of Pseudokirchneriella subcapitata (Harlan, 2010b). The study was conducted in accordance with OECD TG 201 and in compliance with GLP. Loading rates above 0.1 mg/l are above the water solubility of the substance (<0.1 mg/l).

Based on the hydrolysis half-life of the test substance (43 hours at pH 7 and 20-25°C, predicted), and the 96 hour preparation of the test stock solution, it is likely that in the absence of undissolved material the organisms would be exposed mainly to the hydrolysis products of the test substance. For the fish, Daphnia and algae studies, test solutions were prepared above the limit of water solubility of the parent substance. Undissolved material might have been present in the test solutions as a film on the surface or as hydrolysis resistant micelles of the parent substance and oligomers in the water body. Tests with fish and Daphnia have been conducted in filtered and unfiltered test media. The observations during these tests indicate that higher toxicity is observed in unfiltered test media. Consequently, effects are associated with undissoved test material. However, a filter is not able to effectively retain undissolved monomers and oligomers, therefore it is possible that the test organisms in all the tests could have been exposed to undissolved material. Therefore the results should be treated with caution.

The results are supported by a reliable study with the read-across substance triethoxy(octyl)silane (CAS 2943-75-1). A 72-hour EC50 value of >0.13 mg/l and a NOEC value of ≥0.13 mg/l have been determined for the effects of the test substance on growth rate and biomass (yield) of Pseudokirchneriella subcapitata (Springborn Smithers, 2008c). The test was carried out in accordance with OECD TG 201, and in compliance with GLP. Based on the hydrolysis half-life of the test substance (30 hours at pH 7 and 20-25°C, predicted), it is likely that the test organisms were exposed to a mixture of the parent substance and its hydrolysis products.

Refer to the discussion in the IUCLID Section 6 endpoint summary or Section 7.0 of the CSR for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.