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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:
2008-04-29 to 2008-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Radioactivity in samples was measured daily using liquid scintillation counting (LSC). Samples were collected from each test vessel at mid-depth of the test solutions. Samples (10 mL) were collected using a 10-mL volumetric pipette and transferred to a 20-mL glass scintillation vial.
Vehicle:
yes
Details on test solutions:
The nominal test concentrations were selected based on the reported solubility of the test article (6.74 μg/L). The nominal test concentrations were: 0.42, 0.84, 1.7, 3.4 and 6.7 μg/L. A primary stock was prepared by isotopic dilution of the 14C-L4 with non-radiolabelled L4. Approximately 2 mL of DMF (dimethylformamide) was transferred to a 10-mL conical vial with a septum cap. A 7.0 μL aliquot of 14C-L4 (0.00660 g) and a 37.0 μL aliquot of L4 (0.03167 g) was added to the vial through the septum cap. The total weight of 14C-L4 and L4 was 0.03827 g. The contents of the vial were transferred to a 500-mL volumetric flask, brought to volume with DMF, and mixed well giving a final concentration of 77 mg/L. Three 0.05 mL aliquots of the primary stock were added to 10 mL of scintillation cocktail and counted by LSC. The average of the 3 aliquots was 413, 154 dpm. The specific activity of the primary stock was calculated as 48.6 μCi/mg.

Four additional stock solutions were prepared at concentrations of 39, 20, 10 and 5 mg/L by serial dilution of the primary stock with DMF. DMF (0.1 mL/L only was used for the solvent control. To prepare the test solutions, 0.5 mL of the appropriate stock was added to 5 L of algal assay medium (AAM) in a 5-L volumetric flask. The flask was then inverted to mix. Approximately 300 mL of test solution was added to each of the thirteen
replicates and the media blank for each treatment group. Remaining test solution in the volumetric flask was used for initial water quality measurements.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source
Test organisms were obtained from in-house cultures. The source of the original brood stock was strain UTEX 1648, from the University of Texas at Austin, The Culture Collection of Algae, Botany Department, Austin, Texas, in March 1998. The identity of the species was verified by the supplier.

Organism Holding Conditions
Algal cultures were maintained at 24 ± 2 °C on a rotary shaker set at approximately 100 rpm, under continuous cool white fluorescent lighting at a range of 400 ±100 foot candles (4300 ± 1075 lux). The cultures were periodically transferred axenically to new AAM.

Test organisms were impartially added to the test vessels by transferring a specific density of algal cells (nominal concentration equal to 5,000 cells/mL) from a test inoculum into each vessel. In addition, the test vessels were indiscriminately positioned daily on 4 shaker tables in an incubator.

Nutrient Medium
Algal Assay Medium was prepared based on ASTM guideline E 1218-04 with two exceptions. The concentration of NaHC03 was increased from 15 mg/L to 300 mg/L to supply needed C02 in a closed bottle system. The pH of the medium was adjusted to 7.0±0.1 as recommended in ISO 14442. All stocks used to prepare the medium were prepared with sterile distilled water and were filter sterilized (0.22 μm). Medium was prepared with commercially available distilled water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
24.1 to 24.4°C
pH:
Measurements of pH at test initiation ranged from 7.1 to 7.4. On day 1 and 2, pH in the flasks that contained algae ranged from 7.0 to 7.4 and from 7.0 to 7.3, respectively. At test termination, pH in the flasks that contained algae ranged from 8.8 to 9.6 and pH of media blanks ranged from 6.9 to 7.2.
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations were 0.42, 0.84, 1.7, 3.4, and 6.7 μg/L with a control and solvent (DMF) control.

Geometric mean measured test concentrations were calculated to be 0.15, 0.33, 0.70, 1.4 and 2.2 μg/L.

Measured test concentrations at test initiation ranged from 64 to 77 percent of nominal. On Day I and 2 measured test concentrations ranged from 38 to 57 percent of nominal and 32 to 45 percent of nominal, respectively. Measured test concentrations at test termination ranged from 14 to 20 percent of nominal.
Details on test conditions:
Test Apparatus
Test vessels were sterile 300-mL biological oxygen demand (BOD) bottles sealed with a glass stopper. Each test vessel contained two glass marbles to enhance mixing of cells. Test vessels were completely filled with test solution without headspace.

Test Conditions
Test vessels were placed in a temperature controlled incubator and positioned on rotary shakers set at approximately 100 rpm. In addition, the incubator was set at a temperature of 24 ± 2 °C and under continuous cool white fluorescent lighting at a range of 412 - 825 foot-candles (4440 - 8880 lux).

Temperature was measured in a beaker of water adjacent to the shakers in the incubator and light readings were recorded at five indiscriminate areas on the shaker tables daily during the test.

Readings of pH were completed on bulk preparations of each treatment group at test initiation. To maintain axenic conditions, pH readings for each test vessel were measured only at termination.

Observations
Samples for cell counts were collected on day 0 for the original culture and test inoculums. Samples for cell counts were also collected from three test vessels of each treatment group at 24, 48 and 72 hours (within I hour). Prior to sampling, test solutions were emptied into 500-mL glass beakers. The inside of the test vessels were rinsed two times with the test solution using a 60 cc syringe. They were shaken vigorously to ensure all algal cells had been removed from the sides of the test vessel. Approximately 1 mL of the test solution was collected and diluted with Isoton® II diluent solution. Dilution volumes were recorded in the raw data. Samples were counted immediately using a Coulter® Counter. Dilution samples of the medium and diluent blanks were counted at 24, 48 and 72 hours to account for any interference of particles.

At test termination, samples were pooled by treatment and visually inspected for atypical cell morphology. Visual examination showed no concentration-dependent changes in shape, colour or size.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 2.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): none reported
Reported statistics and error estimates:
There was insufficient inhibition of growth rate to determine an EC50 value. The NOEC was determined using Dunnett's test.

Table1. Measurement of Test Concentrations

 

Nominal

Concentration (μg/L)

Day 0

Day 1

Day 2

Day 3

Geometric mean (μg/L)*

Negative Control

<LOQ

<LOQ

<LOQ

 

<LOQ

 

-

Solvent Control

<LOQ

<LOQ

 

<LOQ

 

<LOQ

 

-

0.42

0.27

0.16

 

0.17

 

0.06

0.15

 

0.84

0.58

0.34

 

0.38

 

0.17

0.33

 

1.7

1.2

0.88

 

0.77

 

0.28

0.70

 

3.4

2.5

1.9

 

1.2

 

0.65

1.4

 

6.7

5.1

2.6

 

2.1

 

0.91

2.2

 

'The Limit of Quantitation (LOQ) for the analysis was equivalent to 0.042 μg/L.

*The geometric mean was calculated because the deviation from measured initial concentration was not within±20%.

 

Table2. Test results

 

Nominal

Concentration (μg/L)

Mean cell density after 72 hours (cells/mL)

Mean yield after 72 hours (cells/mL)

% inhibition relative to solvent control

Mean growth rate 0-72 hours

% inhibition relative to solvent control

Negative Control

1147978

 

1142978

 

-

1.8119

 

-

Solvent Control

993911

 

988911

 

-

1.7631

 

-

0.42

905156

 

900156

 

9.0

 

1.7327

 

1.7

 

0.84

1004778

 

999778

 

-1.1

 

1.7658

 

-0.2

 

1.7

875311

 

870311

 

12.0

 

1.7208

 

2.4

 

3.4

795022

 

790022

 

20.1*

 

1.6874

 

4.3*

 

6.7

886178

 

881178

 

10.9

 

1.7254

 

2.1

 

*Indicates a significant difference from the solvent control using Dunnett's test(p≤0.05).

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >2.2 μg/L and NOEC of ≥2.2 μg/L have been determined for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata. The results are expressed as geometric mean measured concentrations.

Description of key information

EC50 (72 hour): >2.2 μg/L, growth rate, Pseudokirchneriella subcapitata, read-across from L4.

NOEC: ≥2.2 μg/L, growth rate, Pseudokirchneriella subcapitata, read-across from L4.

Key value for chemical safety assessment

Additional information

A 72 hour EC50 value of >2.2 μg/L and NOEC of ≥2.2 μg/L have been determined for the effects of decamethyltetrasiloxane (L4, CAS 141-62-8) on growth rate of Pseudokirchneriella subcapitata. The results are expressed as geometric mean measured concentrations.