2-tetradecyloxirane, reaction products with boric acid

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2018 to 18 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Range-finding test: Samples of the fresh test preparations were taken on Days 0 and 2 and of the expired test preparations on Days 2 and 5.
- Definitive test: Water samples were taken from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 5, 9, 14, 19, 26 and 30 and from the old or expired media on Days 2, 7, 12, 16, 21, 28 and 33 (Replicates R1 to R4 pooled) for immediate quantitative analysis. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST WATER
- The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Annex 2 (attached).

Test organisms (species):

Pimephales promelas

Details on test organisms:

ANIMAL WELFARE
- The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo Research Limited, Shardlow, UK policy on animal welfare and the requirements of the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo Research Limited, Shardlow, UK Ethical Review Process.
- The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.

TEST SYSTEM
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20-minute dawn and dusk transition periods. In the 7 days preceding the start of the test, the water temperature was controlled at 25 °C with a dissolved oxygen content of greater than or equal to 8.2 mg O2/L. The breeding stock fish were fed frozen brine shrimp.
- Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 14 November 2018 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
- The diet and diluent water were considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

33 d

Hardness:

total hardness approximately 140 mg/L as CaCO3

Test temperature:

23 to 27 °C

pH:

7.3 to 8.3

Dissolved oxygen:

at least at 3.6 mg/L (equivalent to 43% ASV)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

- Range-finding test: nominal concentrations of 1.0, 10 and 100 mg/L prepared using 22, 220 and 2200 mg in 22 L of test water.
- Definitive test: nominal concentration of 100 mg/L prepared using 2200 mg in 22 L of test water.
- Measured boron concentrations of fresh test preparations were determined to lie in the range 0.210 to 0.362 mg/L (equating to total test item concentrations of 10.7 mg/L to 18.3 mg/L).
- Measured boron concentrations of old test preparations were determined to lie in the range 0.201 and 0.414 mg/L (equating to total test item concentrations of 10.2 mg/L to 21.0 mg/L).

Details on test conditions:

PURPOSE OF THE STUDY
- The study report contains a description of the methods used and results obtained to investigate the toxicity, lethal or sub-lethal effects of the test item on freshly hatched larvae of the fathead minnow (Pimephales promelas).
- Fathead minnow is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.
- In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

VALIDATION OF MIXING PERIOD
- Pre-study work was carried out on a different batch of the test item for Envigo Study Numbers: 41203365 to 41203367 (see Annex 3, attached) to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF.
- A WAF with a nominal loading rate of 100 mg/L was prepared, in duplicate, in deionized reverse osmosis water. One preparation was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples of the WAFs taken for Total Organic Carbon analysis. Increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.
- Samples from the range-finding and definitive tests were analysed for the marker component of total boron dissolved in the WAF.

RANGE-FINDING TEST
- The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing freshly fertilized eggs to a series of nominal test concentrations of 1.0, 10 and 100 mg/L loading rate WAF for a period of 15 days.
- Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.
- Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 ml discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions present.
- The test was conducted in 1 L glass vessels each containing approximately 400 mL of test preparation from Day 0 to 6, increasing to approximately 800 mL from Day 7 to 15. Two replicate flasks were used for each control and test concentration.
- Media renewal was conducted by pouring off old test media whilst retaining test organisms in approximately 50 to 100 mL of old test media, prior to making up to volume with fresh test solutions.
- Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods, for a period of 15 days under semi-static test conditions. The test preparations were renewed on Days 0, 2, 5, 7, 9 and 12.
- The control group was maintained under identical conditions but not exposed to the test item.
- The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily.
- The larvae were fed less than 24 hour old brine shrimp nauplii from Day 7 to the end of the test.

DEFINITIVE TEST
- Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a water accommodated fraction of the test item at a nominal loading rate of 100 mg/L for a period of 33 days at a temperature of 23 ºC to 27 °C under semi-static test conditions.

EXPERIMENTAL PROCEDURE FOR THE DEFINITIVE TEST
- A nominal amount of test item (2200 mg) was separately added to the surface of 22 liters of test water to give the 100 mg/L loading rate on each occasion of preparation. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour.
- Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 ml discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions present.
- The control group was maintained under identical conditions but not exposed to the test item.

EXPOSURE CONDITIONS FOR THE DEFINITIVE TEST
- In the definitive test 1 L glass vessels were used from Day 0 to Day 13 and from Day 14 to the end of the test 5 liter glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 6, 800 mL from Day 7 to Day 13 and 4000 mL from Day 14 to Day 33. Four replicate flasks were used for each control and test concentration.
- Media renewal was conducted by pouring off old test media whilst retaining test organisms in approximately 50 to 100 mL of old test media. This was then made up to volume with fresh media (Days 2, 5, 7, 9, 12, 14 and 16). From Day 19 until the end of the test, old test media (containing test organisms) was passed through a fine sieve, test organisms were then carefully returned to fresh media.
- A semi-static test regime was employed in the test involving a renewal of the test preparations three times per week from the start of the test.
- Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
- The test vessels were aerated via narrow bore glass tubes from Day 12 onwards. The eggs and larvae were not individually identified.
- The larvae were fed less than 24 hour old special grade brine shrimp nauplii from Day 7 to Day 11, and less than 24 hour old basic grade brine shrimp nauplii from Day 12 to the end of the test.

ASSESSMENTS IN THE DEFINITIVE TEST
- Test organism observations: The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus. At the end of the test, the length and wet weight of each surviving fish was determined.
- Water quality: Dissolved oxygen concentrations and pH were recorded before and after each test media renewal. The water temperature and light intensity were recorded daily throughout the test. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration was measured using a Hach Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in Control Replicate R1 using a Testo temperature logger. The water hardness was measured in the bulk test preparation at the start and in each vessel on termination of the test and was determined using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

VALIDATION CRITERIA
- The dissolved oxygen concentration should be greater than 60% of the Air Saturation Value (ASV) throughout the test (equivalent to 4.9 mg O2/L at 25 ºC).
- The water temperature must not differ by more than ± 1.5°C between test chambers or between successive days at any time during the test.
- Analytical measurement of the test loading rates is compulsory.
- The hatching success rate should be greater than 70% and the post-hatch success i.e. survival, should be greater than 75%.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of eggs hatching and post-hatch survival

Remarks on result:

other: no effect on the survival or growth of the larvae

Duration:

33 d

Dose descriptor:

LL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

other: LL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

LL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

other: LL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

33 d

Dose descriptor:

other: EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

33 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

33 d

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

other: EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

33 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Annex 3, attached) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

RANGE-FINDING TEST
- The numbers of eggs hatched, fish surviving to the end of the range-finding test, cumulative mortalities and average weight of fish surviving to the end of the range-finding test are given in Table 1 (attached).
The results showed there was no difference between the control and any of the nominal loading rates tested in terms of hatching, survival and growth.
- A single fish in the 10 mg/L loading rate test group was observed to have a bent spine from Day 8 until the end of the test. Given that this was not a concentration dependant response, this was considered to be due to normal biological variation and as such had no impact on the outcome of the test.
- Based on this information, a single test concentration of 100 mg/L loading rate WAF was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no adverse reactions to exposure were observed.
- Chemical analysis of the freshly prepared test preparations taken for immediate analysis on Days 0 and 2 showed measured boron concentrations of between 0.296 and 16.4 mg/L. Measured boron concentration of the corresponding aged test preparations on Days 2 and 5 showed measured test concentrations of between 0.460 and 14.4 mg/L indicating that the test item was stable under test conditions (see Annex 4, attached). A measured concentration of boron (0.432 mg/L) was detected in the old media in the control on Day 5; however, no boron was detected in the fresh media from the control on Days 0 and 2 or in the old media from the control on Day 2. Given that the fresh media did not contain boron at concentrations above the Limit of Quantification (LOQ), that a slight increase of measured boron was observed in many of the old media samples, and that there was no effect on the hatching or survival of the test organisms, this observation was considered not to have adversely affected the validity or integrity of the test. The presence of boron in the old media is considered most likely to attributable to leaching of boron from the borosilicate glass beakers which were used in the test and also to the provision of brine shrimp nauplii as a feed for the hatched fish from Day 7 of the range-finding test.

CHEMICAL ANALYSIS OF TEST LOADING RATES IN THE DEFINITIVE TEST
- Analysis of the fresh test preparations on Days 0, 5, 9, 14, 19, 26 and 30 showed measured boron concentrations to be between 0.210 and 0.362 mg/L (equating to total test item concentrations of 10.7 mg/L to 18.3 mg/L). Analysis of the old or expired test preparations on Days 2, 7, 12, 16, 21, 28 and 33 showed measured boron concentrations of between 0.201 and 0.414 mg/L (equating to total test item concentrations of 10.2 to 21.0 mg/L) were obtained (see Annex 4, attached). Measured concentrations of boron (0.0104 to 0.0381 mg/L) were detected in the old media in the control on Days 2, 12, 16, 21, 28 and 33; however, no boron was detected in the fresh media from the control (except on Day 30 – 0.0151 mg/L) or in the old media from the control on Day 5. Given that the fresh media did not contain boron at concentrations above the Limit of Quantification (LOQ), that a slight increase of measured boron was observed in many of the old media samples, and that the was no effect on the hatching or survival of the test organisms, this observation was considered not to have adversely affected the validity or integrity of the test. The presence of boron in the old media is considered most likely to attributable to leaching of boron from the borosilicate glass beakers which were used in the test and also to the provision of brine shrimp nauplii as a feed for the hatched fish from Day 7 of the definitive test.
- The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on the nominal loading rate.

NUMBER OF EGGS HATCHING IN THE DEFINITIVE TEST
- The numbers of dead eggs observed during the definitive test are given in Table 2 (attached).
- The number of dead eggs observed was low throughout the test.
- The start of egg hatching was observed on Day 5 and the completion of hatching was observed on Day 7 of the test. There were no significant mortalities or sub-lethal effects of exposure observed.
- Statistical analysis of the hatching data was carried out for the control and 100 mg/L loading rate WAF test group (see Appendix 1, attached). There were no statistically significant differences (P≥0.05) between the control and 100 mg/L loading rate WAF test group and therefore the NOEL based on the number of eggs hatching was 100 mg/L loading rate WAF.

POST-HATCH SURVIVAL IN THE DEFINITIVE TEST
- The number of dead larvae and hatched (live) larvae observed during the definitive test are given in Table 3 and Table 4 respectively (attached).
- The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.
- There were no significant mortalities or sub-lethal effects of exposure observed.
- Statistical analysis of the post-hatch survival data was carried out for the control and the 100 mg/L loading rate WAF test group (see Appendix 1, attached). There were no statistically significant differences (P≥0.05) between the control and 100 mg/L loading rate WAF test group and therefore the NOEL based on post-hatch survival was 100 mg/L loading rate WAF.
- The percentage of eggs hatched and post-hatch survival are given in Table 5 (attached).

LENGTH AND WEIGHT DATA IN THE DEFINITIVE TEST
- The body length and wet weight data for all fish alive at the end of the test are given in Table 6 (attached).
- Statistical analysis of the fish body length data was carried out for the control and 100 mg/L loading rate test group (see Appendix 1, attached). There were no statistically significant differences (P≥0.05) between the control and 100 mg/L loading rate WAF test group and therefore the NOEL based on body length was 100 mg/L loading rate WAF.
- Statistical analysis of the fish wet weight data was carried out for the control and 100 mg/L loading rate test group (see Appendix 1, attached). There were no statistically significant differences (P≥0.05) between the control and 100 mg/L loading rate WAF test group and therefore the NOEL based on wet weight was 100 mg/L loading rate WAF. Given this information and data assessments above, it was considered that no effect on survival or growth attributable to the test item was observed.

OBSERVATIONS IN THE DEFINITIVE TEST
- There was considered to be no significant increase in the number and frequency of abnormal clinical signs noted between the control and test group fish.
- Abnormal observations were noted in all replicates (both control and test group) but were restricted to a very small number of fish per replicate (one to three fish). The observations recorded (in both the control and test group) were pale fish, curved spine, decreased mobility and small. As a dose related response was not apparent, these observations are considered to be due to defects occurring in a natural population, and were not attributable to exposure to the test item.

VALIDITY CRITERIA IN THE DEFINITIVE TEST
- The following validity criteria were achieved during the test:
(i) Dissolved oxygen: Equal to or greater than 60% ASV (required); Greater than 60% ASV on all days except day 12 where the measured dissolved oxygen was 44% ASV (achieved).
(ii) Water temperature between test chambers: ±1.0 °C (required); ±2.1 °C (achieved).
(iii) Water temperature between successive days: ±1.0 °C (required); ±2.4 °C (achieved).
(iv) Hatching success rate in control vessels: >70% (required); 86% (achieved).
(v) Post-hatch survival in control vessels: >75% (required); 91% (achieved).

WATER QUALITY CRITERIA IN THE DEFINITIVE TEST
- The temperature measurements recorded in Control Replicate R1 by a Testo temperature logger are presented in Figure 1 (attached) and were shown to have been maintained at between 24.1 and 26.4 °C. The temperature recorded in each test vessel before and after each media renewal ranged from 23 °C to 27 °C.
- The dissolved oxygen concentration was maintained at least at 3.6 mg/L (equivalent to 43% ASV) and the pH ranged from 7.3 to 8.3.
- Throughout the test the light intensity was observed to be in the range 446 to 691 Lux.
- The water hardness values were observed to range from 154 to 156 mg/L as CaCO3 at the start of the test and from 154 to 158 mg/L as CaCO3 at termination of the test (see Table 7, attached).
VORTEX DEPTH MEASUREMENTS IN THE DEFINITIVE TEST
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

OBSERVATIONS OF TEST ITEM SOLUBILITY IN THE DEFINITIVE TEST
- Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with test item floating on the surface.
- After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface and dispersed throughout. Examination of the WAF showed undissolved material dispersed throughout and therefore it was considered justifiable to remove the dispersed material by the WAF filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed material.
- After dosing the control and 100 mg/L loading rate were observed to be clear, colorless solutions by visual inspection.

Reported statistics and error estimates:

The ELx/LLx values were estimated by inspection of the data. For the estimation of the No Observed Effect Loading Rate (NOEL), the hatching data and the post-hatch survival data for the control and test group were compared using the Fishers Exact Binomial Test. The length and wet weight data for the control and test group obtained on termination of the test were compared using the Student t-test Procedure. All results were calculated using the ToxRat Professional computer software package (ToxRat). As this study was completed as a “limit test” the Lowest Observed Effect Loading Rate (LOEL) was not determined.

Validity criteria fulfilled:

yes

Conclusions:

The application of the test item to newly fertilized eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

GUIDELINE

A study was performed to assess the effects of the test item on newly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a WAF of the test item for a period of 33 days at a temperature of 23 ºC to 27 °C under semi-static test conditions. The test solutions were renewed three times per week throughout the test. The test item solution was prepared by stirring a nominal loading rate of test item (100 mg/L) in test water using a magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for approximately 1 hour. Microscopic inspection of the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Microscopic observation of the WAF was performed after filtering and showed no micro-dispersions of test item to be present. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

 

RESULTS

Chemical analysis of the test preparations was conducted using ICP-MS analysis for the presence of boron. Analysis of the fresh test preparations on Days 0, 5, 9, 14, 19, 26 and 30 showed measured boron concentrations to be between 0.210 and 0.362 mg/L (equating to total test item concentrations of 10.7 mg/L to 18.3 mg/L). Analysis of the old test preparations on Days 2, 7, 12, 16, 21, 28 and 33 showed measured boron concentrations of between 0.201 and 0.414 mg/L (equating to total test item concentrations of 10.2 to 21.0 mg/L) were obtained. Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on the nominal loading rate.

 

CONCLUSION

The application of the test item to newly fertilized eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

The 33-day LL50 (median Lethal Loading Rate) of the test item to fathead minnow based on nominal loading rates was greater than 100 mg/L loading rate WAF for hatching and post-hatch survival. The 33-day EL50 (Effective Loading Rate) of the test item to fathead minnow based on nominal loading rates was greater than 100 mg/L loading rate WAF length and weight. The No Observed Effect Loading rate was 100 mg/L loading rate WAF

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

A key study was performed to assess the effects of the test item on newly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test.Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a WAF of the test item for a period of 33 days at a temperature of 23 ºC to 27 °C under semi-static test conditions. The test solutions were renewed three times per week throughout the test. The test item solution was prepared by stirring a nominal loading rate of test item (100 mg/L) in test water using a magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for approximately 1 hour. Microscopic inspection of the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Microscopic observation of the WAF was performed after filtering and showed no micro-dispersions of test item to be present. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

 

Chemical analysis of the test preparations was conducted using ICP-MS analysis for the presence of boron. Analysis of the fresh test preparations on Days 0, 5, 9, 14, 19, 26 and 30 showed measured boron concentrations to be between 0.210 and 0.362 mg/L (equating to total test item concentrations of 10.7 mg/L to 18.3 mg/L). Analysis of the old test preparations on Days 2, 7, 12, 16, 21, 28 and 33 showed measured boron concentrations of between 0.201 and 0.414 mg/L (equating to total test item concentrations of 10.2 to 21.0 mg/L) were obtained. Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on the nominal loading rate.The application of the test item to newly fertilized eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.