Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

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:

30 July 2020 to 11 January 2021

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)

Version / remarks:

2013

Deviations:

yes

Remarks:

temperature variations with no impact on results or integrity of the study (see below)

GLP compliance:

yes

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. All samples were taken for analysis on the day of sampling. A duplicate set of samples was taken and stored frozen for further analysis if required.
- 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, 12, 19, 26 and 30 and from the old or expired media on Days 2, 7, 14, 21, 28 and 33 (Replicates R1 to R4 pooled) for quantitative analysis. Samples from Days 0, 2 , 12, 14 and 33 were taken for analysis on the day of sampling whereas the samples taken on Days 5, 7, 19, 21, 26, 26 and 30 were stored frozen prior to analysis. Duplicate samples were taken 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 (Fleck 2750 Duplex Dechlorination unit) 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 were provided in Annex 2 of the full study report.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST SYSTEM
- The test was carried out using newly fertilized eggs of fathead minnows (Pimephales promelas).
- 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 to 26 °C with a dissolved oxygen content of greater than or equal to 6.7 mg O2/L. The breeding stock fish were fed daily with frozen brine shrimp and ZM granular commercial fish food.
- 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 09 December 2020 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 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Hardness:

- hardness 136 to 144 mg/L as CaCO3 (start of test)
- hardness 148 to 178 mg/L as CaCO3 (termination of test)

Test temperature:

23.1 °C to 26.0 °C in test vessels

pH:

7.8 to 8.6

Dissolved oxygen:

at least at 6.38 mg/L (equivalent to 77% ASV at 25 °)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L test item

DEFINITIVE TEST
- Nominal loading rates of 2.2, 4.5, 10, 22 and 45 mg/L test item

Details on test conditions:

PURPOSE OF STUDY
- This study describes methods used and results obtained to investigate the toxicity, lethal or sub-lethal effects of the test item on the early-life stages 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 2019 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
- 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
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE-FINDING TEST
- The loading rates to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing newly fertilized fathead minnow 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 (10, 100 and 1000 mg) were each separately added to the surface of 10 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 3 hours and the mixtures allowed to stand for 1 hour. A 10 mL and a 2.0 mL pipette, covered at one end with Nescofilm, were submerged with the vessels (sealed end down to mid-depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the Nescofilm was removed using the 2.0 ml pipette. On occasions, 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 particles or micro-dispersions were present.
- A control WAF was also prepared as per the test WAF’s but without the addition of test item.
- The test was conducted in 1 L glass vessels each containing approximately 400 mL of test preparation from Day 0 to 7, increasing to approximately 800 mL from Day 7 to 15. Two replicate vessels were used for each control and test concentration.
- 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 with 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 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 freshly hatched brine shrimp nauplii from Day 7 to Day 14.

INITIAL TEST
- An initial test was conducted following the same method as for the definitive test; however, this test was terminated on Day 12 as a result of significant mortalities which did not follow a dose response. No explanation can be provided for the observed mortalities and therefore the test was restarted using the same range of concentrations.

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 water accommodated fractions of the test item at a nominal loading rates of 2.2, 4.5, 10, 22 and 45 mg/L for a period of 33 days at a temperature of 25 ºC under semi- static test conditions.
- On Days 0, 2, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28 and 30. Nominal amounts of test item (44, 90, 200, 440 and 900 mg) were each separately added to the surface of 20 L of test water to give the 2.2, 4.5, 10, 22 and 45 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 3 hours and the mixtures allowed to stand for 1 hour. A 10 mL and a 2.0 mL pipette, covered at one end with Nescofilm, were submerged with the vessels (sealed end down to mid-depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the Nescofilm was removed using the 2.0 ml pipette. The WAF removed by mid-depth siphoning (the first 75 to 100 ml discarded) to give the 2.2, 4.5, 10, 22 and 45 mg/L loading rate WAFs.
- A control WAF was also prepared as per the test WAF’s but without the addition of test item.
- The control group was maintained under identical conditions but not exposed to the test item.
- In the definitive test 1 L glass vessels were used from Day 0 to Day 17 and from Day 17 to the end of the test 5 L glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 7, 800 mL from Day 7 to Day 17 and 4000 mL from Day 17 to Day 33. Four replicate flasks were used for each control and test concentration.
- A semi-static test regime was employed in the test involving a renewal of the test preparations three times per week to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.
- 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 with 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 8 onwards. The eggs and larvae were not individually identified.
- The larvae were fed freshly hatched brine shrimp nauplii from Day 7 of the test.

WATER QUALITY CRITERIA
- Dissolved oxygen concentrations, water temperature and pH were recorded at the beginning and end of the test and before and after each test media renewal. Light intensity was 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 were measured using a Hach Flexi handheld meter and the temperature was measured using a digital thermometer. The temperature was also monitored approximately every hour in a surrogate vessel 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 results of the test are considered valid if the following criteria are met:
(i) The dissolved oxygen concentration must be between 60 % and 100 % of the Air Saturation Value (ASV) throughout the test.
(ii) 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 and should be within the range 25 ± 1.5 °C.
(iii) Concentrations of the test item in solution should be maintained within ± 20 % of the mean measured values.
(iv) The hatching success rate should be greater than 70 % and the post-hatch success i.e. survival, should be greater than 75 %.

MAJOR COMPUTERIZED SYSTEMS
- Building management: Delta control system (Version 3.4.0)
- Temperature logger: Testo Comfort Software Basic Version 5.0
- Professional computer software package: ToxRat (Version 3.3.0)
- Electronic communication system: Veeva QMS

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

LL10

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

other: LL20

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

LL10

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: post-hatch survival

Duration:

33 d

Dose descriptor:

other: LL20

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: post-hatch survival

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: post-hatch survival

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: post-hatch survival

Duration:

33 d

Dose descriptor:

EL10

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: body length

Duration:

33 d

Dose descriptor:

other: EL20

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: body length

Duration:

33 d

Dose descriptor:

EL50

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: body length

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: body length

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: body length

Duration:

33 d

Dose descriptor:

EL10

Effect conc.:

26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: wet weight

Remarks on result:

other: 95 % confidence limits 11 mg/L to 62 mg/L loading rate WAF

Duration:

33 d

Dose descriptor:

other: EL20

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: wet weight

Duration:

33 d

Dose descriptor:

EL50

Effect conc.:

> 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: wet weight

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: wet weight

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: wet weight

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 3 hours. Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 3 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 were given in Table 1 of the full study report.
- The results showed there was no difference between the control and test concentrations of 1.0 and 10 mg/L loading rate WAF in terms of hatching, survival and growth, however, differences were at a test concentration of 100 mg/L loading rate WAF where 100% mortality of the exposed fish occurred during the exposure period.
- No sub-lethal effects were observed in the control and at the test concentration of 1.0 mg/L loading rate WAF, however, sub-lethal effects were observed at 10 mg/L loading rate WAF. These sub-lethal effects observed were abnormal swimming and pale.
- Based on this information test concentrations of 2.2, 4.5, 10, 22 and 45 mg/L loading rate WAF were selected for the definitive test.
- Chemical analysis of the fresh test preparations on Day 0 showed measured boron concentrations (corrected for boron concentrations in the control media) at 10 and 100 mg/L loading rates were 0.064 and 0.96 mg/L, corresponding to test item concentrations of 5.4 and 81 mg/L. Chemical analysis of the old test preparations on Day 2 showed corrected measured boron concentrations at 10 and 100 mg/L loading rates were 0.074 and 0.10 mg boron/L, corresponding to test item concentrations of 6.3 and 84 mg/L. Measured concentrations from the 1.0 mg/L loading rate were below the limit of quantification.
- Chemical analysis of the fresh test preparations on Day 2 showed a probable sample mix up had occurred. The 10 and 100 mg/L samples both returned results of less than the limit of quantification, whereas the control and 1.0 mg/L samples provided measured boron concentrations of 0.77 and 0.71 mg/L, corresponding to test item concentrations of 6.5 and 60 mg/L. Chemical analysis of the old test preparations on Day 5 showed corrected measured boron concentrations at 10 and 100 mg/L loading rates were 0.077 and 0.67 mg boron/L, corresponding to test item concentrations of 6.4 and 57 mg/L, providing evidence that the Day 2 fresh samples had been mixed up. Measured concentrations from the 1.0 mg/L loading rate were below the limit of quantification.

DEFINITIVE TEST – CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the fresh test preparations on Days 0, 5, 12, 19, 26 and 30 (see Annex 4) showed measured boron concentrations (corrected for boron concentrations in the control media) ranged from less than the limit of quantification to 0.461 mg boron/L, corresponding to test item concentrations of less than the limit of quantification and 39 mg/L.
- Chemical analysis of the old test preparations on Days 2, 7, 14, 21, 28 and 33 showed corrected measured boron concentrations ranged from less than the limit of quantification to 0.458 mg boron/L, corresponding to less than the limit of quantification and 39 mg/L. The limit of quantification for boron was determined to be 0.12 mg/L corresponding to test item concentration of 10 mg/L.
- 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 nominal loading rates only.

DEFINITIVE TEST – NUMBER OF EGGS HATCHING
- The numbers of dead eggs observed during the definitive test were given in Table 2 of the full study report. The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.
- The start of egg hatching was observed on Day 5 of the test and completion of hatching was observed on Day 7 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations. It was not possible to calculate 95 % confidence limits for the LL50 values as the data generated did not fit the models available for the calculation of confidence limits.
- Statistical analysis of the hatching data was carried out for the control and all loading rates. There were no statistically significant differences (P ≥ 0.05), between the control and all test groups and therefore the NOEL based on the number of eggs hatching was 45 mg/L loading rate WAF. Correspondingly the LOEL based on the number of eggs hatching was not determined.

DEFINITIVE TEST – POST-HATCH SURVIVAL
- The number of dead larvae and hatched (live) larvae observed during the definitive test were given in Table 3 and Table 4 of the full study report respectively. 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 in any of the test concentrations. It was not possible to calculate 95 % confidence limits for the LL50 values as the data generated did not fit the models available for the calculation of confidence limits.
- Statistical analysis of the post-hatch survival data was carried out for the control and all loading rates. There were no statistically significant differences (P ≥ 0.05), between the control and all test groups and therefore the NOEL based on post-hatch survival was 45 mg/L loading rate WAF. Correspondingly the LOEL based on post-hatch survival was not determined.
- The percentage of eggs hatched and post-hatch survival were given in Table 5 of the full study report.

DEFINITIVE TEST – LENGTH AND WEIGHT DATA
- The body length and wet weight data for all fish alive at the end of the test were given in Table 6 of the full study report. The data showed a 3 % reduction in attained body length was observed at the test concentrations of 22 and 45 mg/L. Given that no effects in excess of 10 % were observed, it was not possible to calculate any ELx values. It was not possible to calculate 95 % confidence limits for the EL50 values as the data generated did not fit the models available for the calculation of confidence limits.
- The relationship between body length and concentration at Day 33 (28 days post-hatch) was given in Figure 1 of the full study report. The relationship between body weight and concentration at Day 33 (28 days post-hatch) was given in Figure 2 of the full study report.
- Statistical analysis of the fish body length data was carried out for the control and all loading rates. There were no statistically significant differences (P ≥ 0.05), between the control, 2.2, 4.5 and 10 mg/L loading rate WAF test groups, however all other loading rates were significantly different (P < 0.05) and, therefore the NOEL based on body length was 10 mg/L loading rate WAF. Correspondingly the LOEL based on body length was 22 mg/L loading rate WAF.
- Statistical analysis of the fish wet weight data was carried out for the control and all loading rates. There were no statistically significant (P ≥ 0.05) differences between the control, 2.2, 4.5 and 10 mg/L loading rate WAF, however all other loading rates were significantly different (P < 0.05) and, therefore the NOEL based on wet weight was 10 mg/L loading rate WAF. Correspondingly the LOEL based on wet weight was 22 mg/L loading rate WAF.

VALIDATION CRITERIA
- Validity criteria achieved during the test were as shown in the table below.

WATER QUALITY CRITERIA
- The temperature measurements recorded in a surrogate test vessel incubated alongside the test vessels by a Testo temperature logger were presented in Figure 3 of the full study report and were shown to have been maintained at between 24.2 and 25.5 °C. The temperature recorded in each test vessel before and after each media renewal ranged from 23.1 °C to 26.0 °C.
- The dissolved oxygen concentration was maintained at least at 6.38 mg/L (equivalent to 77 % ASV at 25 °) and the pH ranged from 7.8 to 8.6. Throughout the test the light intensity was observed to be in the range 509 to 626 Lux.
- The water hardness values presented in Table 8 of the full study report were observed to range from 136 to 144 mg/L as CaCO3 at the start of the test and from 148 to 178 mg/L as CaCO3 at termination of the test.

VORTEX DEPTH MEASUREMENTS
- 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.

OBSERVATION OF TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- After dosing all control and test concentrations were observed to be clear, colorless solutions by visual inspection.

Reported statistics and error estimates:

- EL/LLx Calculations: No statistically significant concentration responses were determined for hatching and post-hatch survival data; however, LLx values were determined by Probit analysis using Linear Maximum-Likelihood regression. ELx values for length and weight of fish surviving at the end of the test were estimated using the 3-Parameter Normal Cumulative Distribution Function using the ToxRat computer software package (ToxRat Solutions GMBH) and were confirmed by inspection of the data.
- Lowest Observed Effect Loading Rate and No Observed Effect Loading Rate Determinations: For the estimation of the Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL) the hatching data and the post-hatch survival data for the control and each test group were compared using the Chi2 2x2 Table Test with Bonferroni Correction. The length and wet weight data for the control and each test group obtained on termination of the test were compared using Williams Multiple Sequential t-test incorporating Trend analysis by contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution. All results were calculated using the ToxRat computer software package (ToxRat Solutions GMBH).

VALIDATION CRITERIA

Criterion	Required	Achieved
Dissolved oxygen	Equal to or greater than 60 % ASV (equivalent to 4.9 mg O2/L at 25 °C)	Lowest measured value was 6.4 mg O2/L (equivalent to 77 % ASV)
Water temperature between test chambers	± 1.5 °C	± 1.7 °C
Water temperature between successive days	± 1.5 °C	± 2.1 °C
Hatching success rate in control vessels	> 70 %	91 %
Post-hatch survival in control vessels	> 75 %	95 %

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal test concentrations, exposure of the early-life stages of fathead minnow to the test item gave EL50 (33 d) > 45 mg/L when body length and wet weight were considered. The corresponding No Observed Effect Loading Rate was 10 mg/L. The Lowest Observed Effect Loading Rate was reported as 22 mg/L.

Executive summary:

GUIDELINE

A study was performed to assess the effects of the test item on the early life stages 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 (four replicates of 20 eggs per group) were exposed to WAFs of the test item for a period of 33 days at a temperature of 23 to 26 ºC under semi-static test conditions. The test solutions were renewed three times per week throughout the test. The test item solutions were prepared by stirring a nominal loading rates of test item (2.2, 4.5, 10, 22 and 45 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 for 3 hours. After the stirring period the mixtures allowed to stand for 1 hour before the aqueous phases or WAFs were removed by mid-depth siphoning to give the 2.2, 4.5, 10, 22 and 45 mg/L loading rate WAFs.

 

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 fresh test preparations on Days 0, 5, 12, 19, 26 and 30 showed measured boron concentrations (corrected for boron concentrations in the control media) ranged from less than the limit of quantification to 0.461 mg boron/L, corresponding to test item concentrations of less than the limit of quantification and 39 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14, 21, 28 and 33 showed corrected measured boron concentrations ranged from less than the limit of quantification to 0.458 mg boron/L, corresponding of less than the limit of quantification and 39 mg/L. The limit of quantification for boron was determined to be 0.12 mg/L corresponding to test item concentration of 10 mg/L.

 

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 nominal loading rates only.

 

CONCLUSION

Exposure of the early-life stages of fathead minnow to the test item gave the following results based on the nominal test concentrations:

Parameter	Endpoint	Concentration (mg/L loading rate WAF)
Number hatched	LL10	> 45
Number hatched	LL20	> 45
Number hatched	LL50	> 45
Number hatched	No Observed Effect Loading Rate	45
Number hatched	Lowest Observed Effect Loading Rate	Not determined
Post-hatch survival	LL10	> 45
Post-hatch survival	LL20	> 45
Post-hatch survival	LL50	> 45
Post-hatch survival	No Observed Effect Loading Rate	45
Post-hatch survival	Lowest Observed Effect Loading Rate	Not determined
Body length	EL10	> 45
Body length	EL20	> 45
Body length	EL50	> 45
Body length	No Observed Effect Loading Rate	10
Body length	Lowest Observed Effect Loading Rate	22
Wet weight	EL10	26 (95% confidence limits 11 to 62)
Wet weight	EL20	> 45
Wet weight	EL50	> 45
Wet weight	No Observed Effect Loading Rate	10
Wet weight	Lowest Observed Effect Loading Rate	22

Description of key information

Based on nominal test concentrations, exposure of the early-life stages of fathead minnow to the test item gave EL50 (33 d) > 45 mg/L when body length and wet weight were considered. The corresponding No Observed Effect Loading Rate was 10 mg/L. The Lowest Observed Effect Loading Rate was reported as 22 mg/L (OECD 210).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Additional information

GUIDELINE

A study was performed to assess the effects of the test item on the early life stages 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 (four replicates of 20 eggs per group) were exposed to WAFs of the test item for a period of 33 days at a temperature of 23 to 26 ºC under semi-static test conditions. The test solutions were renewed three times per week throughout the test. The test item solutions were prepared by stirring a nominal loading rates of test item (2.2, 4.5, 10, 22 and 45 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 for 3 hours. After the stirring period the mixtures allowed to stand for 1 hour before the aqueous phases or WAFs were removed by mid-depth siphoning to give the 2.2, 4.5, 10, 22 and 45 mg/L loading rate WAFs.

 

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 fresh test preparations on Days 0, 5, 12, 19, 26 and 30 showed measured boron concentrations (corrected for boron concentrations in the control media) ranged from less than the limit of quantification to 0.461 mg boron/L, corresponding to test item concentrations of less than the limit of quantification and 39 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14, 21, 28 and 33 showed corrected measured boron concentrations ranged from less than the limit of quantification to 0.458 mg boron/L, corresponding of less than the limit of quantification and 39 mg/L. The limit of quantification for boron was determined to be 0.12 mg/L corresponding to test item concentration of 10 mg/L.

 

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 nominal loading rates only.

 

CONCLUSION

Exposure of the early-life stages of fathead minnow to the test item gave the following results based on the nominal test concentrations:

Parameter	Endpoint	Concentration (mg/L loading rate WAF)
Number hatched	LL10	> 45
Number hatched	LL20	> 45
Number hatched	LL50	> 45
Number hatched	No Observed Effect Loading Rate	45
Number hatched	Lowest Observed Effect Loading Rate	Not determined
Post-hatch survival	LL10	> 45
Post-hatch survival	LL20	> 45
Post-hatch survival	LL50	> 45
Post-hatch survival	No Observed Effect Loading Rate	45
Post-hatch survival	Lowest Observed Effect Loading Rate	Not determined
Body length	EL10	> 45
Body length	EL20	> 45
Body length	EL50	> 45
Body length	No Observed Effect Loading Rate	10
Body length	Lowest Observed Effect Loading Rate	22
Wet weight	EL10	26 (95% confidence limits 11 to 62)
Wet weight	EL20	> 45
Wet weight	EL50	> 45
Wet weight	No Observed Effect Loading Rate	10
Wet weight	Lowest Observed Effect Loading Rate	22