Iodine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-08-05 to 2010-08-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted April 4, 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Description: Bluish-black prills with metallic luster
Batch: 182827052
Purity: 99.8%
Test substance storage: At room temperature in the dark
Expiry date: 29 December 2010

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

First experiment was conducted at 0.010, 0.032, 0.10, 0.32, 1, 10, 100 and 1000 mg/L. The concentrations at and below 1 mg/L were prepared using a stock solution. The stock solution was prepared by adding 5.08 mg test substance to 1000 mL of Milli-RO water (tap water purified by reverse osmosis). Ultrasonic dispersion and thorough mixing (34 minutes) was used to accelerate dissolution and to ensure homogeneity. An amount of synthetic sewage feed (16 mL), activated sludge (200 mL) and an adequate amount of the test substance stock were mixed and made up to 500 mL with Milli-RO water in a 1-litre bottle.
Due to the low water solubility of lodine, the use of a stock solution was not possible for the three highest concentrations. These concentrations were prepared separately in Milli-RO water. 1-Litre test bottles were filled with 250 mL of test substance mixtures with initial loading rates of 20, 200 and 2000 mg/L, respectively. The mixtures were stirred in closed dark brown glass bottles for 26-26.5 hours. Subsequently, 16 mL synthetic sewage feed, 200 mL sludge and Milli-RO water up to 500 mL were added resulting in loading rates of 10, 100 and 1000 mg/L.
Since significant inhibition was found at a loading rate of 1000 mg/L, a second experiment was performed with loading rates from 100 to 1000 mg/L, forming a geometric progression with a factor 1.8. In this experiment all concentrations were prepared separately in Milli-RO water. 1-Litre test bottles were filled with 250 mL of test substance mixtures with initial loading rates of 200, 360, 640, 1120 and 2000 mg/L, respectively. The mixtures were stirred in closed dark brown glass bottles for 25-26 hours. Subsequently, 16 mL synthetic sewage feed, 200 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates.
Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The test system was micro-organisms in activated sludge. The source of the test organisms is activated sludge that has been freshly obtained from a municipal sewage treatment plant (Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands), receiving predominantly domestic sewage.

In terms of preparation of the sludge, this was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.6 and 3.7 g/L of sludge, as used in experiment 1 and 2, respectively). The pH was 8.2 and 7.6 on the day of testing in experiment 1 and 2, respectively. The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

The composition of the medium (Adjusted ISO medium), formulated using tap-water purified by reverse osmosis is as follows:

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

As iodine is known as a water disinfectant, the test was initiated applying a 3 hour contact time. Aeration and stirring took place during the contact time.

Test temperature:

The temperature of the test medium was between 18.1 and 19.1°C and between 18.4 and 18.9°C in experiment 1 and 2, respectively.

pH:

Experiment 1: 8.3-8.7
Experiment 2: 6.7-8.0

Dissolved oxygen:

Experiment 1: 7.3-8.9 mg O2/L at the start
Experiment 2: 7.8-9.3 mg O2/L at the start

Nominal and measured concentrations:

Experiment 1: 0.010, 0.032, 0.10, 0.32, 1, 10, 100 and 1000 mg/L (loading rate)
Experiment 2: 100, 180, 320, 560, 1000 mg/L (loading rate)

Details on test conditions:

- Contact time: 3 hours, during which aeration and stirring took place.
- Vessels: AII glass, approximately 300 mL oxygen bottles and 1-litre test bottles.
- Air supply: Clean. oil-free air.
- Performance of the test:
In case of the use of a stock solution: the synthetic sewage feed (16 mL), activated sludge (200 mL) and an adequate amount of the test substance stock were mixed and made up to 500 mL with Milli-RO water in a 1-litre bottle.
In case of testing loading rates: following the overnight stirring period, the synthetic sewage feed (16 mL) and the activated sludge (200 mL) were added to the test substance mixture. MiIIi-RO water was added to provide a final volume of 500 mL with a final test substance loading of 100 mg/L. The mixture was aerated during the contact time, using a pipette as an aeration device.
After the 3-hour contact time, a well mixed sample of the content was poured into a 300 mL oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.
This procedure was repeated for all concentrations of the test substance. Two controls without test substance were tested in each test series, one at the start and one at the end.

Reference substance (positive control):

yes

Remarks:

The batch of activated sludge was checked for sensitivity by using the reference substance 3,5-dichlorophenol (CAS Number: 591-35-5).

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI - 120-670 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI - 40-290 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

570 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI: 230-1430 mg/L

Details on results:

The test met acceptability criteria in that:
1. The difference between the controls was within 15% of each other.
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (10.8 and 9.8 mg/L in experiment 1 and 2, respectively).

In the first experiment no significant inhibition of respiration rate of the sludge was recorded at and below 100 mg/L, whereas 92% inhibition was recorded at a loading rate of 1000 mg/L. In the second experiment the inhibitory effect of Iodine on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from a 12% inhibition at a loading rate of 100 mg/L to 98% inhibition at a loading rate of 1000 mg/L. The calculated percentages inhibition allowed for a reliable determination of EC-values.

The respiration rate from each vessel, in mg O2/L/hour was calculated from the linear part of the respiration curve. The inhibitory effect (percentage inhibition) was calculated. The percentage inhibition was plotted against the logarithm of the calculations. The EC50 was determined using linear regression analysis and was expressed as 95% confidence limits.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (10.8 and 9.8 mg/L in experiment 1 and 2, respectively).

Validity criteria fulfilled:

yes

Conclusions:

The EC10 for test item (3 hours contact time) is 110 mg/L and the EC50 (3 hours contact time) is 280 mg/L.

Executive summary:

This study investigated the ability of test item to adversely affect aerobic microbial treatment plants. The source of test organisms is activated sludge freshly obtained from a municipal sewage treatment plant receiving predominanty domestic sewage. As this study was conducted to OECD Guideline 209: Activated Sludge, Respiration Inhibition Test this is considered to meet the Klimisch reliability criteria of 1 - reliable without restriction. The EC10 for test item (3 hours contact time) is 110 mg/L and the EC50 (3 hours contact time) is 280 mg/L.

Description of key information

The EC10 for test item (3 hours contact time) is 110 mg/L and the EC50 (3 hours contact time) is 280 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

280 mg/L

EC10 or NOEC for microorganisms:

110 mg/L

Additional information

This study investigated the ability of test item to adversely affect aerobic microbial treatment plants. The source of test organisms is activated sludge freshly obtained from a municipal sewage treatment plant receiving predominanty domestic sewage. As this study was conducted to OECD Guideline 209: Activated Sludge, Respiration Inhibition Test this is considered to meet the Klimisch reliability criteria of 1 - reliable without restriction. The EC10 for test item (3 hours contact time) is 110 mg/L and the EC50 (3 hours contact time) is 280 mg/L