Registration Dossier

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:
2011-09-15 to 2011-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Method: Prior to test initiation, a sample was collected from the primary stock prepared in DMF. Additionally, stability and concentration verification was determined daily using GC/MS. Samples were collected from one analytical replicate for each treatment group. An additional analytical replicate containing the test medium without algae was sampled from the 0.32 mg/L treatment group on Days 1, 2, and 3.
Vehicle:
yes
Details on test solutions:
The nominal test concentrations were 0.020, 0.040, 0.080, 0.16 and 0.32 mg/L. A primary stock was prepared by adding 0.323 g of the test substance to a 100-mL volumetric flask (stock concentration 3.23 mg/mL). The flask was then brought to volume with DMF. Four secondary stock solutions were prepared at concentrations of 0.20, 0.40, 0.80 and 1.6 mg/mL by 50% serial dilutions 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, below the surface. The flask was then mixed using a sterile stir bar and placed on a stir plate for approximately 5 minutes. Test vessels were filled approximately half way with the appropriate test solution. For each concentration, the replicates requiring algae were inoculated with approximately 5,000 cells. Each test vessel was then filled completely without headspace and sealed with a glass stopper. All test solutions were observed to be clear and colorless indicating that the test solutions were homogeneous.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): 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.

- Method of cultivation: 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+/-1076 lux). The cultures were periodically transferred axenically to new media.

ACCLIMATION

- Culturing media and conditions (same as test or not): Same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.8 to 24.1°C
pH:
7.0 to 7.5 at test initiation

7.3 to 9.4 at the end of the test
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 0(Control), 0.020, 0.040, 0.080, 0.16 and 0.32 mg/L

Geometric mean measured test concentrations in treated vessels: 0.0064, 0.015, 0.029, 0.061 and 0.12 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 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 labelled with the study number, test concentration and replicate.
- Type (delete if not applicable): closed
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Static
- Initial cells density: 5000 cells/mL
- Control end cells density: 718644 cells/mL

TEST DESIGN
The controls and the 0.16, 0.080, 0.040, and 0.020 mg/L utilized thirteen replicates; four of those vessels were used for analytical sampling and contained test medium plus algae. Cell counts were not conducted on those four replicates. A media blank containing test medium without algae was also prepared for each test concentration and control for use with the Coulter® Counter.

In order to determine whether the presence of algae had an impact on the test concentrations, an additional replicate of the 0.32 mg/L treatment group was prepared for Days 1, 2, and 3. Seven of the 16 replicates were used for analytical sampling; four with test medium plus algae and three with test medium only. Cell counts were not conducted on those seven replicates.

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


GROWTH MEDIUM
- Growth medium: Algal Assay Medium (AAM) was prepared based on ASTM guideline E 1218-04, with two exceptions: 1) The concentration of NaHCO3 was increased from 15 mg/L to 300 mg/L to supply needed CO2 in a closed bottle system. The pH of the medium was adjusted to 7.0 ± 0.1 as recommended in ISO 14442 [8]. All stocks used to prepare the medium were prepared with sterile Milli-Q water and were filter sterilized (0.22 m). Medium was prepared with Milli-Q water dispensed from the Millipore® Milli-Q Integral Water Purification System which is analyzed annually for contaminants. No contaminants have been shown to be present which might adversely impact the viability of the cultures or study.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water dispensed from the Millipore® Milli-Q Integral Water Purification System
- Culture medium different from test medium: No
- Intervals of water quality measurement: 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. Measurements 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples for cell counts were collected on Day 0 for the original culture and test inoculum. Samples for cell counts were also collected from three test vessels of each treatment group at 24, 48 and 72 hours (within 1 hour). 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, color or size


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
EC50 values for yield (EyCx) and growth rate (ErCx) at 72 hours were estimated by visual examination of the data.

The no observed effect concentration (NOEC) values were determined for growth rate and yield at 72 hours using William’s test. All statistical analyses were performed using TOXSTAT Version 3.5 software.

Table 1. Results of analysis of test media

Nominal

Concentration

(mg/L)

 

 

Day 0

 

 

Day 1

 

 

Day 2

 

 

Day 3

Geometric Mean (mg/L)2

Percent of Nominal

Negative Control

 

BLQ1

BLQ

BLQ

BLQ

--

--

Solvent Control

BLQ

BLQ

BLQ

BLQ

--

--

 

0.020

 

0.011

0.0080

0.0063

0.0029

0.0064

32

 

0.040

 

0.025

0.020

0.016

0.0061

0.015

37

 

0.080

 

0.051

0.039

0.032

0.011

0.029

37

 

0.16

 

0.11

0.080

0.064

0.025

0.061

38

 

0.32 (with algae)

 

0.18

0.16

0.12

0.055

0.12

37

0.32 (without algae)

NA3

0.17

0.14

0.12

0.15

47

1BLQ = Below Limit of Quantitation (LOQ for the analysis was equivalent to 0.0000725 mg/L).

2The geometric mean was calculated because the deviation from the measured initial concentration was not within±20% [2].

3NA = Not Applicable. No analysis at test initiation. Geometric mean was calculated using the 0.32 mg/L (with algae) Day 0 value.  

Table 2. Test results - mean cell densities (cells/mL)

Geometric Mean Measured Concentration

(mg/L)

0-h

24-h

48-h

72-h

Control

5000

14849

81871

718644

Solvent Control

5000

17680

77924

605289

0.0064

5000

11200

67887

818600

0.015

5000

13236

64262

847133

0.029

5000

16642

60224

811844

0.061

5000

12864

52380

823289

0.12

5000

9756

45867

584200

Table 3. Test results - mean yield (cells/mL)

Geometric Mean Measured

Concentration

(mg/L)

72 hours

% Inhibition**

Negative Control

713644

-19

Solvent Control

600289

0

0.0064

813600

-36

0.015

842133

-40

0.029

806844

-34

0.061

818289

-36

0.12

579200

3.5

**Percent inhibition was calculated in comparison to the solvent control.

Table 4. Test results - mean growth rates

Geometric Mean Measured

Concentration (mg/L)

0 - 72 hours

% Inhibition**

Negative Control

1.6556

-3.6

Solvent Control

1.5984

0

0.0064

1.6987

-6.3

0.015

1.7101

-7.0

0.029

1.6965

-6.1

0.061

1.7009

-6.4

0.12

1.5830

1.0

**Percent inhibition was calculated in comparison to the solvent control.

Validity criteria fulfilled:
yes
Conclusions:
A 72 hour EC50 value of >0.12 mg/L and a NOEC of ≥0.12 mg/L have been determined for the effects of the registered substance on growth rate of Pseudokirchneriella subcapitata. The results are based on geometric mean measured exposure concentrations.

Description of key information

Short-term toxicity to algae: 72-h ErC50 >0.12 mg/l and NOEC >=0.12 mg/l (measured) (highest concentration tested) (OECD guideline 201)

Key value for chemical safety assessment

Additional information

A 72 hour EC50 value of >0.12 mg/l and NOEC of 0.12 mg/l (geometric mean measured concentration) (highest concentration tested) have been determined for the effects of the registration substance on growth rate of Pseudokirchneriella subcapitata (Dow Corning Corporation, 2011). In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the tested substance. Effectively these results indicate no effects at the limit of achievable solubility in test medium.