Iodine

Toxicity to aquatic algae and cyanobacteria

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:

From 2010-07-30 to 2010-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Description: black pellets
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Vehicle:

no

Details on test solutions:

The test item is slightly soluble in water and pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. It was therefore considered appropriate to prepare the test item using a saturated solution method of preparation.

An amount of test item (200 mg) was dispersed in 1 litre of culture medium with the aid of high shear mixing at approximately 7500 rpm for 30 minutes. After mixing any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 10, 3.2, 1.0, 0.32 and 0.10% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 8.1 mL of algal suspension to give the required test concentrations of 0.10, 0.32 , 1.0, 3.2 and 10% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Desmodesmus subspicatus (previously known as Scenedesmus subspicatus) is a freshwater unicellular alga representative of a wide variety of natural habitats, and is therefore considered as an important non-target organism in freshwater ecosystems. Desmodesmus subspicatus Strain CCAP 276/20 were obtained from the Culture Collection of Algae and Protozoa (CCAP) Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 ºC. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 1E+04 - 1E+05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 degrees C for control and test groups.

pH:

The pH values of the control cultures were observed to range from pH 7.6 – 7.8 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. The pH values in the test concentrations ranged from 7.2 - 7.7.

Nominal and measured concentrations:

Nominal: 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution
Measured concentrations at 0-Hour: 0.025, 0.077, 0.25, 0.74 and 2.6 mg/L (three replicate flasks per concentration)

Details on test conditions:

- Exposure conditions:
Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test item at 0-Hour measured concentrations of 0.025, 0.077, 0.25, 0.74 and 2.6 mg/L for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 degrees C. Six flasks (250 mL glass conical flasks) each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.48 E+05 cells per mL. Inoculation of 500 mL of test medium with 8.1 mL of this algal suspension gave an initial nominal cell density of 4 E+03 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1℃ under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

- Culture medium:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The composition is specified below.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Range finding study:
Test concentrations: 0.010, 0.10, 1.0, 10 and 100% v/v saturated solution
Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.010 and 0.10% v/v saturated solution. However, growth was observed to be reduced at 1.0, 10 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as the reference item

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits: 0.11 - 0.16 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Verification of test concentrations:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.025 to 2.6 mg/L. Analysis of the test preparations at 72 hours showed measured test concentrations to range from 92% to 98% of the 0-Hour measured test concentrations with the exception of the 2.6 mg/L test concentration which saw a decline to 76% of the 0-Hour measured test concentration. Given that this was the highest test concentration employed which was far in excess of the EC50 values it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only.

- Validation criteria:
The cell concentration of the control cultures increased by a factor of 31 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 3.94 E+03 cells per mL
Mean cell density of control at 72 hours : 1.22 E+05 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 30% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

- Observations on cultures:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.025, 0.077 and 0.25 mg/L, however no intact cells were observed to be present in the test cultures at 0.74 and 2.6 mg/L.

- Observations on test item solubility:
At the start of the test all control, 0.025, 0.077 and 0.25 mg/L test cultures were observed to be clear colourless solutions. The 0.74 mg/L test cultures were observed to be extremely pale yellow solutions whilst the 2.6 mg/L test cultures were observed to be very pale yellow solutions. After the 72-Hour test period all control and 0.025 mg/L test cultures were observed to be green dispersions. The 0.077 mg/L test cultures were observed to be very pale green dispersions, the 0.25 mg/L test cultures were observed to be extremely pale green dispersions, the 0.74 mg/L test cultures were observed to be extremely pale yellow solutions and the 2.6 mg/L test cultures were observed to be very pale yellow solutions.

- Growth data:
Based on the study results it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test item over the 72 Hour exposure period.
Inhibition of growth rate:
ErC10 (0 - 72 h): 0.042 mg/L
ErC20 (0 - 72 h): 0.065 mg/L
ErC50 (0 - 72 h): 0.13 mg/L; 95% confidence limits 0.11 - 0.16 mg/L
LOEC(growth rate): 0.077 mg/L
Inhibifion of yield:
EyC10 (0 - 72 h): 0.060 mg/L
EyC20 (0 - 72 h): 0.064 mg/L
EyC50 (0 - 72 h): 0.073 mg/L; 95% confidence limits 0.070 - 0.076 mg/L
LOEC(yield): 0.077 mg/L

Results with reference substance (positive control):

The EC50 and NOEC for growth rate is presented below:
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L; ErC50 (0 – 72 h) : 0.74 mg/L

It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

For each individual test vessel, percentage inhibition (arithmetic axis) is plotted against test concentraton (logarithmic axis) and a line fitted by eye or by computerised interpolation. Any data points that were identified as outliers in the first phase of data evaluation were disregarded when fitting the concentration response curve. The EC50 values were read from the graph. 95% confidence limits were calculatd using the simplified method of Litchfield and Wilcoxon (1949).

Reference in protocol: Litchfield and Wilcoxon (1949) J Pharmacol Exp Ther 96, 99-113

The No Observed Effect Concentration (NOEC) is the test concentration immediately below the Lowest Observed Effect Concentration (LOEC) which when compared to the control, has no statistically significant effect (P ≥ 0.05) with a given exposure time. One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 for growth rate was 0.13 mg/L and the NOEC for growth rate was 0.025 mg/L.

Executive summary:

This 72 hour study investigated the aquatic toxicity of test item to algae (Desmodesmus subspicatus). As this study was conducted to OECD Guideline 201: Alga, Growth Inhibition Test, this is considered to meet the Klimisch reliability criteria of 1 - reliable without restriction. The results were based on the 0-Hour measured test concentrations only as the highest test concentration saw a decline to 76% of the 0-Hour measured test concentration. Given that this was the highest test concentration employed which was far in excess of the EC50 values it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only. Based on the calculations, the 72 hour EC50 for growth rate was 0.13 mg/L and the NOEC for growth rate was 0.025 mg/L.

Description of key information

Toxicity to aquatic algae: EC50 (72 hours): 0.13 mg/L; NOEC (72 hours): 0.025 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.13 mg/L

EC10 or NOEC for freshwater algae:

0.025 mg/L

Additional information

The reviewed 72 hour study investigated the aquatic toxicity of test item to algae (Desmodesmus subspicatus). As this study was conducted to OECD Guideline 201: Alga, Growth Inhibition Test, this is considered to meet the Klimisch reliability criteria of 1 - reliable without restriction. The results were based on the 0-Hour measured test concentrations only as the highest test concentration saw a decline to 76% of the 0-Hour measured test concentration. Given that this was the highest test concentration employed which was far in excess of the EC50 values it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only. Based on the calculations, the 72 hour EC50 for growth rate was 0.13 mg/L and the NOEC for growth rate was 0.025 mg/L.