Iodine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The methodology and results presented below for iodine (I2) have been included as part of a well documented publication that investigated the toxicity of iodine to Daphnia magna and Rainbow Trout (Oncorhynchus mykiss ). The testing protocol follows the Ontario Ministry of the Environment testing guidelines, Canada. A couple of deviations were reported in the publication compared with these guidelines. The deviations relate to the use of hydroponic plant food to grow algae fed to Daphnia and the supplementation of selenium to the culture water. The use of hydroponic plant food instead of Bristol's solution to assist the growth of algae is not considered likely to significantly affect the culture procedure. Also, the addition of selenium to the culture water is beneficial at the concentration used, as a deficiency can result in the poor health of daphnids. Information in the Biological Test Method: Acute Lethality Test Using Daphnia spp. (Environment Canada, 1990) states that "insufficient waterborne selenium may cause deterioration of the cuticle of daphnids, shorter life, and failure of progeny to mature and reproduce". The information from this publication has also been compared with OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test) to further assess the reliability and validity of this study. The test is considered to meet the validity criteria in OECD Guideline 202. Although no results or observations were stated for the control group in the testing regime, it is assumed that no mortalities occurred. This assumption is based on the results from the preliminary testing where the maximum concentration of ethanol (used to dissolve iodine) to which the Daphnia were exposed was 0.005%. This test confirmed that any toxicity in the main study was due to iodine as ethanol did not cause lethality in Daphnia. In terms of the dissolved oxygen concentration, this was 10.1 mg/L in the Standard Winnipeg River Treatment which was determined to change minimally during the experiment. The dissolved oxygen concentration under conditions of high water hardness (Milner Ridge Spring) was also 10.1 mg/L and also changed minimally during the experiment. These concentrations are greater than the minimum level stated in the validity criteria, ≥3 mg/l. A number of other aspects in this publication are also compliant with this guideline: - Healthy populations of daphnia magna were used as the test species, and the animals used for testing were less than 24 hours old. - The water hardness (as CaCO3) used in the culture water for Daphnia magna (Milner Ridge Spring) was 151 ± 6 mg/L and the concentration under conditions of high water hardness was 222 ± 1 mg/L (as CaCO3). Both of these values fall within the recommended hardness range for Daphnia magna, as specified in OECD Guidelines. - The pH values in culture water (Milner Ridge Spring), standard Winnipeg treatment and Milner Ridge Spring water are 8.2 ± 1, 8.1 ± 1 and 8.3 ± 1 are within the pH range according to OECD guidelines. - The temperature 20°C (±2°C) and light-controlled (16-h light, 8-h dark) environment are consistent with OECD guidelines. - The test was not aerated. - The tests were monitored at 24 and 48 hours. - pH and dissolved oxygen (and also conductivity and total iodine concentration) were measured for each test solution at the beginning and end of the study. - The deviation between the final and the initial ITOT concentrations is less than 20%. - The endpoints have been calculated to 95% confidence limits. There are also factors that are however, not consistent with OECD Guideline 202: - Compliance to GLP is not stated in the publication although it is possible that this study was conducted according GLP principles as a suitable test guideline was adhered to. - Detailed information on the test substance such as impurities was not provided. - The toxicity endpoint for the Daphnia study is stated as an LC50 in that death of the test species is defined as no visible internal movement. In OECD Guideline 202, the EC50 is based on immobilisation (after gentle agitation, those animals that are not able to swim within 15 seconds). However, both of these endpoints are investigating the same effect (i.e. no movement) so the difference in terminology is not considered to affect the reliability of the results. - The water hardness in the standard Winnipeg river treatment was 45.9 ± 0.5 (as CaCO3) which falls outside the range specified in OECD Guidelines, 140 - 250 mg/L (as CaCO3). However, the high water hardness sample (Milner Ridge Spring) in the culture and dilution water adheres to this range, where the values are 151 ± 6 mg/L and 222 ± 1 mg/L. The LC50s of both the standard sample (Winnipeg river treatment) and the high water hardness sample have therefore been considered in this endpoint study record. - It is not stated if there was an acclimatisation period before the start of the study and whether the Daphnia culture conditions (light, temperature) are comparable to the test conditions. - No reference substance was used as a means of assuring the test conditions are reliable, but this does not appear to be a requirement in OECD Guideline 202, in that the guidelines state that a reference substance "may be tested...". - A total of 10 neonates were assessed for each test concentration, the minimum recommended number in OECD Guideline 202 is 20 animals. The main deficiency with this study are the number of neonates used (10/concentration) compared with that recommended in OECD Guideline 202. However, the use of 10 neonates is consistent with Canadian guidelines (Environment Canada, 1990) where ≥ 10 daphnids are recommended per concentration. As this number is consistent with national guidelines, the study is still considered as reliable. Furthermore, the study also meets the validity criteria as specified in OECD Guideline 202. In summary, whilst there are some deficiencies in the study and the information reported, these are not considered to significantly affect the overall reliability or validity of this study which is broadly comparable with OECD Guideline 202: Daphnia sp., Acute Immobilisation Test. An overall reliability score of 2 (reliable with restrictions) is considered appropriate. Reference: Environment Canada (July 1990 with May 1996 amendments): Environmental Protection Series. Biological Test Method: Acute Lethality Test Using Daphnia spp. Report EPS 1/RM/11 http://www.etc-cte.ec.gc.ca/organization/bmd/pubs/pubs_en/1RM11EnglishFinal.pdf

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Ontario Ministry of the Environment protocol. Poirier DG, Westlake GF, Abernethy SG (1988) Daphnia magna acute lethality toxicity test protocol. Ontario Ministry of the Environment, Toronto, Ontario, Canada

Deviations:

yes

Remarks:

Rather than using Bristol's solution as suggested in the guidelines a commercially available two-part hydroponic plant food was used. The culture water for Daphnia was supplemented with Se to a final concentration of approximately 1.0 μg Se/L

GLP compliance:

not specified

Remarks:

No data has been selected as the publication does not state GLP compliance. However, it is possible that this study was conducted according to the principles of GLP as a test guideline was adhered to.

Analytical monitoring:

yes

Details on sampling:

The initial chemical analyses on I2 for both the rainbow trout and the Daphnia, were performed on the trout subsample only. Initial chemical analyses for pH, conductivity, dissolved oxygen, and ITOT were performed on a sample aliquot of each test solution. Upon completion of the toxicity tests, the five 30-mL aliquots of each test solution were pooled and subsampled for final chemical analyses. No adjustments were made to replace the loss of iodine from either the Daphnia test solutions during the test period.

As part of the publication, nine water varieties were compared to explore the toxicity modifying role of water chemistry. All dilution waters were sampled prior to use and analyzed for total iodine (ITOT), Na+, K+,Ca2+, Mg2+ , CI-, SO4 2 -, alkalinity (as CaCO3), pH, hardness (as CaCO3), CuTOT, PbTOT, NiTOT, CdTOT, ZnTOT, FeTOT, MnTOT, electrical conductivity, total organic and inorganic carbon (TOC, TIC), and dissolved oxygen (D.O.).

Vehicle:

yes

Details on test solutions:

The iodine test solutions were prepared from elemental iodine I2 (99%, Sigma, St Louis, MO).

Preliminary testing determined that the lethal (LC50) iodine concentrations were 10.0 mg I/L as elemental iodine (I2).

A control (0%) and six concentrations of iodine (1, 3, 10, 30, 60, 100% of the preliminary lethal concentrations) were used for each test. The 10.0 mg I/L as I2 was identical for both the Daphnia and the trout, so the test solutions for the Daphnia tests were subsampled from the trout test solutions.

For I2, a stock solution (20 g I/L) was prepared by dissolving 2.0 g of elemental iodine into 100 mL of ethyl alcohol (Ethanol, 99.9%, J.T. Baker, Winnipeg, MB). A measured volume of this stock solution was added to obtain the desired test concentration. No adjustments were made to replace the loss of iodine from either the Daphnia test solutions during the test period. No reference study was used in this test. Preliminary testing exposed Daphnia to equivalent concentration ranges of ethanol to confirm that toxicity was attributable to iodine. The maximum concentration of ethanol to which the Daphnia were exposed was 0.005%. This maximum concentration was also non-lethal.

Test organisms (species):

Daphnia magna

Details on test organisms:

Stocks of Daphnia magna were raised in the laboratory following a minor modification to the Ontario Ministry of the Environment protocol. The Daphnia were fed a mixture of two algal species: Selenastrum capricornutum (UTCC #37) and Chlorella emersonii (UTCC #86) both of which were obtained as pure cultures from the University of Toronto Algal Culture Collection of Algae and Cyanobacteria. The algal cultures were grown in a nutrient medium under artificial lighting. Rather than using Bristol's solution as suggested in the Ontario Ministry of the Environment protocol, a commercially available two-part hydroponic plant food ("Bumper crop," Rambridge Structure and Design Limited, Calgary, AB) was used. In addition, to prevent Se-deficiency, the culture water for Daphnia was supplemented with Se to a final concentration of approximately 1.0 µg Se/L. The use of hydroponic plant food instead of Bristol's solution to assist the growth of algae is not considered likely to siginificantly affect the culture procedure. The addition of selenium to the culture water is beneficial at the concentration used, as a deficiency can result in the poor health of daphnids. Information in the Biological Test Method: Acute Lethality Test Using Daphnia spp. (Environment Canada, 1990) states that "insufficient waterborne selenium may cause deterioration of the cuticle of daphnids, shorter life, and failure of progeny to mature and reproduce" based on publications cited in this method. It is not stated regarding whether there was an acclimatisation period before the start of study and whether the Daphnia culture conditions (light, temperature) are comparable to the test conditions.

The culture water supply was local springwater collected from an artesian aquifer in a glacial moraine, and filtered through activated charcoal, crushed limestone, and polyester floss prior to use. A summary of the chemistry of the Daphnia culture water is found in Table l. (note - this is the same table number as used in the Laverock et al. 1995 publication.)

Daphnia in various stages of growth were maintained to provide healthy populations and a steady supply of neonates. One day prior to testing, all neonates were removed from the tanks to ensure that only animals that were ≤ 24 hours old were used for testing. The testing age of the neonates is consistent with OECD Guideline 202.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

There was no post exposure observation period.

Hardness:

Refer to Table 2 for a summary of the water chemistry used in the toxicity tests in this publication (this is the table number in the publication and is also included further below in this endpoint study record). The hardness for the standard water sample (Winnipeg River treatment) is 45.9 ± 0.5 mg/L. According to OECD Guideline 202, hardness between 140 and 250 mg/L (as CaCO3) is recommended for Daphnia magna. Therefore the Daphnia test data on Daphnia magna conducted under conditions of high water hardness (Milner Ridge Spring) is also presented. The hardness in the Milner Ridge Spring sample is 222 ± 1 mg/L which adheres to the range in the OECD Guidelines.

Test temperature:

The 48-h static non-aerated toxicity tests were conducted at 20°C (±2°C) in a temperature controlled water bath.

pH:

Refer to Table 2 for a summary of the water chemistry used in the toxicity tests in this publication. The pH for the standard water sample (Winnipeg River treatment) is 8.1 ± 0.1. The pH in the high water hardness sample (Milner Ridge Spring) is 8.3 ± 0.1. Both pH values adhere to the range in the OECD Guidelines.

Dissolved oxygen:

Refer to Table 2 for a summary of the water chemistry used in the toxicity tests in this publication. The dissolved oxygen content for the standard water sample (Winnipeg River treatment) and high water hardness sample is 10.1 mg/L.

Salinity:

Refer to Table 2 for a summary of the water chemistry used in the toxicity tests in this publication. The chloride content for the standard water sample (Winnipeg River treatment) is 2.60 ± 0.13 mg/L. The chloride content in the high water hardness sample (Milner Ridge Spring) is 13.2 ± 1.3 mg/L.

Nominal and measured concentrations:

The concentrations used in this study were a control (0%) and six concentrations of iodine (1, 3, 10, 30, 60, 100% of the preliminary lethal concentrations. For iodine, these concentrations correspond to 0.1, 0.3, 1, 3, 6 and 10 mg I/L.

The measured (initial) iodine concentrations were compared to the expected concentrations, and similarly, the final ITOT concentrations were compared with the initial concentrations. The results showed that little iodine was lost from solutions during the test period. The mean ratio of the final ITOT to the initial ITOT concentrations was 0.94 of the initial concentrations of I2. This ratio means that the deviation between the final and the initial ITOT concentrations is less than 20% which is in accordance with OECD Guideline 202.

Details on test conditions:

The 48-h static, non-aerated toxicity tests were conducted at 20°C (±2°C) in a temperature controlled water bath and a light-controlled (16-h light, 8-h dark) environment. These conditions are consistent with OECD Guideline 202. However, it is not stated regarding whether there was an acclimitisation period before the start of study and whether the Daphnia culture conditions (light, temperature) are comparable to the test conditions.

Five test tubes for each toxicant concentration were labelled and filled with 30-mL of test solution. Each set of tubes was placed into a water bath. Once the test solutions had reached 20°C (±2°C) two Daphnia neonates were placed into each test tube so that a total of ten neonates were tested at each toxicant concentration. The use of ten neonates is not consistent with OECD Guideline 202, where it is stated that 20 animals are recommended at each concentration. However, 10 neonates is recommended in Canadian guidelines, noting that this publication originated from Canada. As the number of neonates used is consistent with national guidelines, the study is still considered as reliable.

The time and date were noted. The tests were monitored at 24 and 48-h. These monitoring times are consistent with OECD Guideline 202. At these same intervals, the mortality in each test solution was monitored. Death was defined as no visible internal movement. The endpoint used for this study was an LC50 rather than an EC50, as defined in OECD Guideline 202. In this guideline, the EC50 is based on immobilisation (after gentle agitation, those animals that are not able to swim within 15 seconds). However, both of these endpoints are investigating the same effect (i.e. no movement) so the difference in terminology is not considered to affect the reliability of the results.

Reference substance (positive control):

not required

Remarks:

A positive control is not required in OECD Guideline 202, in that the guidelines state that a reference substance "may be tested for EC50 as a means of assuring that the test conditions are reliable".

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Iodine data: The 95% confidence range for this LC50 ranged between 0.51- 0.68 mg/L. This LC50 value was for the Standard Winnipeg sample.

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Iodine data: The 95% confidence range for this LC50 ranged between 0.30 - 1.00 mg/L. This LC50 value was for the high water hardness (Milner Ridge Spring) sample.

Details on results:

The most sensitive and relevant data endpoint for iodine has been included in the effect concentrations section above. The full results table from the publication is included below.

As stated under the "nominal and measured concentrations" field, little iodine was lost from solutions during the test period. The mean ratio of the final ITOT to the initial ITOT concentrations was 0.94 of the initial concentrations of I2

Routine measurements of temperature, dissolved oxygen, pH, and electrical conductivity also indicated that the test conditions changed minimally during the experiment. The monitoring of pH and dissolved oxygen at the beginning and the end of the study is consistent with OECD Guideline 202. The temperature remained within the specifications of 20 ± 2°C in the Daphnia test. The dissolved oxygen concentrations were routinely near the atmospheric saturation value in all test containers. No results or observations were stated for the control group, but it is assumed that no mortalities occurred. This assumption is based on the results from the preliminary testing where the maximum concentration of ethanol (used to dissolve iodine) to which the Daphnia were exposed was 0.005%. This test confirmed that any toxicity in the main study was due to iodine as ethanol did not cause lethality in Daphnia.

The raw data is not presented, although this information would not be expected in a publication. Observations in the test groups were also not provided.

The chemical toxicity (LC50) of I2 to Daphnia in the standard Winnipeg River treatment was calculated at 0.59 mg/L.

The increased water hardness from 7.5 to 45.9 mg/L reduced the toxicity of I2, although a further increase of water hardness to 222 mg/L had no additional effect. In the high water hardness test the LC50 was 0.55 mg/L for iodine. With respect to the effect of Cl- concentrations, elemental iodine was most toxic at low Cl- concentrations. High concentrations of TOC had no effect on the toxicity of I2 to Daphnia.

Results with reference substance (positive control):

A positive control is not required in OECD Guideline 202.

Reported statistics and error estimates:

The LC50 values for each test series were calculated using a toxicity database system (Toxdata Version 2.0) issued by the Ontario Ministry of the Environment. Most calculations were completed using the Spearman-Karber method included in this database. A further description of this method is unavailable, and the raw data and calculations for deriving the LC50 are not available in the publication. This method is a non parametric statistical procedure for estimating the LC50 and the associated 95% confidence interval.
(United States Environmental Protection Agency, 2009: Short-Term Methods For Estimating the Chronic Toxicity of Effluents and Receiving Water to Marine and Estuarine Organisms- Appendix I Spearman-Karber Method. URL: http://www.epa.gov/waterscience/methods/wet/disk1/ctmapg-l.pdf)

The most sensitive and relevant data endpoint for iodine has been included in the effect concentrations section above. The full results table from the publication is included below.

Median lethal toxic concentration (LC₅₀) of iodine toDaphnia magna. All LC₅₀values were calculated using the Spearman-Karber method (extracted from publication)

 

Iodine Species	Standard Winnipeg River treatment (a)	Hardness (b)		Chloride (c)		Total organic carbon (c)
		Low (7.5)	High (222)	Medium (13)	High (61)	Medium (7.8)	High (28.2)
I2(mg/L)	0.59 (0.51 – 0.68) (d)	0.16 (0.14 – 0.19)	0.55 (0.30 – 1.00)	1.75 (1.54 – 1.99)	1.73 (1.0-3.0)	0.85 (0.55 – 1.32)	0.59 (0.51 – 0.68)

 

(a) Standard Winnipeg River treatment (charcoal filtered) had medium hardness (45.9 mg/L as CaCO₃), low TOC (2.7 mg/L), and low chloride (2.6 mg/L) concentrations

(b) mg/L as CaCO₃

(c) mg/L

(d) 95% confidence ranges

 

Validity criteria fulfilled:

yes

Remarks:

The information from this publication (conducted to the Ontario Ministry of the Environment testing guidelines) has been compared with OECD Guideline 202. Refer to field "Rationale for reliability including deficiencies" for further details.

Conclusions:

The 48 hour LC50 for iodine was 0.59 mg/L (standard Winnipeg River treatment) and 0.55 mg/L (high water hardness sample; Milner Ridge Spring) The LC50 using the high water hardness sample is selected as the endpoint for this study as the water hardness of the culture and dilution water adheres to the range in OECD Guideline 202.

Executive summary:

The publication by Laverock, MJ Stephenson, M and Macdonald CR (1995) investigated the toxicity of iodine to Daphnia magna and Rainbow Trout (Oncorhynchus mykiss).

This endpoint study record reviews the short term toxicity test (Daphnia magna) using iodine (I2) as the test substance. With respect to iodine, Daphnia magna were exposed to 0.1, 0.3, 1, 3, 6 and 10 mg I/L for 48 hours under static, non aerated conditions. The study also investigated whether the chemistry of the water used in the studies affected the toxicity results. The test guidelines cited for the conduct of the study were the Ontario Ministry of the Environment Guidelines. The information reported in the publication is broadly comparable with OECD Guideline 202 Daphnia sp., Acute Immobilisation Test. Although there are some deficiencies in the study and the information reported, these are not considered to significantly affect the overall reliability or validity of this study. An overall reliability score of 2 (reliable with restrictions) is considered appropriate.

The chemical toxicity (LC50) of I2 to Daphnia in the standard Winnipeg River treatment was calculated at 0.59 mg/L (95% confidence range: 0.51 - 0.68 mg/L). The calculation of this endpoint to 95% confidence limits is consistent with OECD Guideline 202. The increased water hardness from 7.5 to 45.9 mg/L reduced the toxicity of I2. Although a further increase of water hardness to 222 mg/L had no additional effect. In the high water hardness test the LC50 was 0.55 mg/L (95% confidence range: 0.30 - 1.00 mg/L). With respect to the effect of Cl- concentrations, iodine was most toxic at low Cl- concentrations.

In terms of the validity of the study, although no results or observations were stated for the control group, a dilution of ethanol (used to dissolve iodine) did not result in lethality in preliminary testing. This test confirmed that any toxicity in the main study was due to iodine as ethanol did not cause lethality in Daphnia. With regards to the dissolved oxygen concentration, this was 10.1 mg/L in the Standard Winnipeg River Treatment which was determined to change minimally during the experiment. The dissolved oxygen concentration under conditions of high water hardness (Milner Ridge Spring) was also 10.1 mg/L and also changed minimally during the experiment. These concentrations are greater than the minimum level stated in the validity criteria, ≥3 mg/L. The results for elemental iodine are therefore considered to be valid.

The 48 hour LC50 for I2 (elemental iodine) is 0.59 mg/L (standard Winnipeg River treatment) and 0.55 mg/L (high water hardness sample; Milner Ridge Spring) The LC50 using the high water hardness sample is selected as the endpoint for this study as the water hardness of the culture and dilution water adheres to the range in OECD Guideline 202.

 

Description of key information

Short term toxicity to aquatic invertebrates:

LC50 (48 hours): 0.55 mg/L (Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.55 mg/L

Additional information

One short term toxicity study to aquatic invertebrates (Daphnia magna) has been identified for iodine. This is a 48 hour study under static, non aerated conditions that investigated the toxicity of iodine to Daphnia magna (toxicity to rainbow trout was also separately investigated in the same publication). [Laverock, MJ Stephenson, M and Macdonald CR (1995)]. The study also investigated whether the chemistry of the water used in the studies affected the toxicity results.

The test guidelines cited for the conduct of the study were the Ontario Ministry of the Environment Guidelines. The information reported in the publication is broadly comparable with OECD Guideline 202 Daphnia sp., Acute Immobilisation Test. Although there are some deficiencies in the study and the information reported, these are not considered to significantly affect the overall reliability or validity of this study. An overall reliability score of 2 (reliable with restrictions) is considered appropriate.

The 48 hour LC50 for I2 (elemental iodine) is 0.59 mg/L (standard Winnipeg River treatment) and 0.55 mg/L (high water hardness sample; Milner Ridge Spring) The LC50 using the high water hardness sample is selected as the endpoint for this study as the water hardness of the culture and dilution water adheres to the range in OECD Guideline 202.

Reference:

Laverock MJ, Stephenson M, and Macdonald CR (1995): Toxicity of Iodine, Iodide and Iodate to Daphnia magna and Rainbow Trout (Oncorhynchus mykiss ) Arch. Environ. Contam. Toxicol. 29, 344 -350