Pidolic acid

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November 2016 to 13 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

Adopted 17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- Lot Number: 20140124
- Purity; 99.92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item will be stored at ambient temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): dissolved in water.
- Concentrations; 4.27, 9.39, 20.7, 45.5 and 100 mg.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- as a dissolved solution.

Analytical monitoring:

yes

Details on sampling:

The concentration of (2S)-5-oxopyrrolidine-2-carboxylic acid was determined using a liquid
chromatography with UV detection (LC-UV) method of analysis. Three replicate 6 mL
samples (duplicate samples for analysis with one sample held in reserve) were taken from
the control and the test solutions at 0 hours, from the fresh test solutions at 24 and 72 hours
and from the aged test solutions at 24, 72 and 96 hours. All replicates were stored frozen on
the day of sampling until required for analysis.

Vehicle:

yes

Details on test solutions:

The test solutions were made up by the direct addition of (2S)-5-oxopyrrolidine-2-
carboxylic acid to 5 L of dilution water in the test vessels. Each test solution was then made
up to 15 L with dilution water ensuring the test item was in solution and well mixed. The
test solutions were renewed at the 24 and 72 hour time points and the fish were transferred
to the fresh test solutions with a minimal amount of aged test solution using a net.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: Oncorhynchus mykiss
- Source: supplied by Allenbrook Trout Farm, East Dorset, BH21 5LT, UK
- Length at study initiation (length definition, mean, range and SD): 4.8 cm (4.0 -5.9 cm)
- Weight at study initiation (mean and range, SD): 1.09 g (0.41-2.21 g).

ACCLIMATION
-The fish stock had been maintained in aerated dilution water and similar environmental conditions to the bioassay for at least 12 days prior to the start of the bioassay.
- Fish were not fed 24 hours before the start or during the bioassay.
- Health during acclimation (any mortality observed): Mortality was 2% in the fish used for the study during the seven days prior to the start of the definitive bioassay.

FEEDING DURING TEST (as applicable)
- The stock were fed daily using a commercial trout feed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

To verify actual exposure levels, analysis of (2S)-5-oxopyrrolidine-2-carboxylic acid was carried out on the freshly prepared test solutions at 0, 24 and 72 hours and the aged test solutions at 24, 72 and 96 hours.

Post exposure observation period:

Observations for sub-lethal effects and mortality in the control and test item groups were
conducted at approximately 15 minutes and 1, 2, 3, 4, 5, 24, 48, 72 and 96 hours exposure.

Hardness:

The system was re-filled using a mixture of carbon filtered (de-chlorinated) and reverse osmosis water to maintain a total hardness of between 10 and 250 mg CaCO3/L.

Test temperature:

The study was maintained at 15 ± 2ºC

pH:

A pH of between 6.0 and 8.5.

Dissolved oxygen:

the dissolved oxygen (DO) concentration was maintained above 60% air saturation value (ASV) for the study duration.

Nominal and measured concentrations:

Nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. All measured concentrations were maintained within 80 to 120%.

Details on test conditions:

TEST SYSTEM
- Test vessel: The test vessels were all glass aquaria each filled with 15 L of the test solution. This gave a loading rate of 0.5 g (wet weight fish)/L. The test vessels were provided with constant aeration from the main laboratory air supply. The tanks were covered with plastic lids, to prevent the test media becoming contaminated.

TEST MEDIUM / WATER PARAMETERS
- The dilution water used for the bioassays and holding of the fish stocks was from the same source. The source was the laboratory freshwater supply from a re-circulating filtration system which undergoes at least 30 % weekly water changes. The system was re-filled using a mixture of carbon filtered (de-chlorinated) and reverse osmosis water to maintain a total hardness of between 10 and 250 mg CaCO3/L and a pH of between 6.0 and 8.5. The bioassay was conducted under semi-static conditions with test solution renewals at 24 and 72 hours.

REPLICATION
- For the range-finding and definitive bioassays, no replication was used for the control and each test concentration. Each test vessel contained 3 fish in the range-finding bioassay and 7 fish in the definitive bioassay.

OTHER TEST CONDITIONS
- The definitive bioassay was conducted under semi-static conditions with constant aeration and no adjustment of the pH throughout the duration. A 16: 8 hour light: dark photoperiod was maintained with a 1 hour dawn dusk period at the start and end of the cycle. The study was maintained at 15 ± 2ºC and temperature was monitored continuously in a surrogate vessel using IceSpy electronic environmental equipment (Skye Instruments, Powys, UK). Water quality measurements (including pH, dissolved oxygen and temperature) were performed at 0 hours, at 24 and 72 hours on the fresh and aged test solutions and at 96 hours. The pH was maintained within the range 6.0 - 8.5 and the dissolved oxygen (DO) concentration was maintained above 60% air saturation value (ASV) for the study duration.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Individual vessels each containing 7 fish were exposed for a period of 96 hours. Observations were conducted at 15 minutes and 1, 2, 3, 4, 5, 24, 48, 72 and 96 hours exposure. The results of any additional
observations conducted were added to the nearest allocated time point.
- Mortality assessments of individual fish were assessed at approximately 15 minutes, 1, 2, 3, 4, 5, 24, 48, 72 and 96 hours. Other observations were made at additional time points; and were allocated to the nearest 24 hour time point. Sub-lethal observations of visible abnormalities and abnormal behaviour (e.g. loss of equilibrium, abnormal swimming behaviour) or physiological (pigmentation change, respiratory function) were also noted at observation points.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on the results from the range-finding bioassay and consultation with the Study Monitor, the definitive bioassay was performed at nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L (The test item purity was assumed to be 100% for the preparation the treatment solutions as the Certificate of Analysis was not available prior to the start of the definitive bioassay)
- Justification for using less concentrations than requested by guideline:
- Range finding study: A range-finding bioassay was conducted to establish the concentration range which would provide toxicity in the definitive bioassay if possible. Individual vessels containing 3 fish were exposed to control and nominal treatment levels of 0.01, 0.1, 1.0, 10 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. The test item purity was assumed to be 100% for the preparation the treatment solutions. Mortality and sub-lethal effects were assessed after 15 minutes and 1, 2, 3, 4, 5, 6, 24, 48, 72 and 96 hours

STATISTICAL ANALYSIS
Statistical analysis of the 96 hour survival data was performed (using ToxCalc v.5.023, 1999). Analysis was performed using the nominal (2S)-5-oxopyrrolidine-2-carboxylic acid treatment concentrations.
The Fisher’s Exact hypothesis test (2 tail, p>0.05) was used to test for any significant differences between to the control and treatment concentrations and to determine a NOEC and LOEC for fish mortality.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

After 96 hours exposure no mortality was observed at nominal concentrations of 4.27, 9.39, 20.7, 45.5 or 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. No mortality occurred in the control.
Sub-lethal effects including hyperventilation, surface swimming, darkened pigmentation, lethargy and loss of orientation were observed in the 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L.

Sublethal observations / clinical signs:

Table 5 Sub-lethal Effect Observations

Nominal Concentration (mg (2S)-5- oxopyrrolidine-2- carboxylic acid /L)	Number affected (initial population = 7 fish)
	15 mins	1 hour	2 hours	3 hours	4 hours	5 hours	24 hours	48 hours	72 hours	96 hours
Control	-	-	-	-	-	-	-	-	-	1 DK
4.27	-	-	-	-	-	-	-	-	-	-
9.39	-	-	-	-	-	-	-	-	-	-
20.7	-	-	-	-	-	-	-	-	-	-
45.5	7 HV	7 HV	4 HV	2 HV	2 HV	3 HV	7 HV	1 HV	7 HV 1 SS 2 DK	2 HV 1 SS 2 DK
100	7 HV 3 SS	7 HV 2 SS	7 HV	7 HV	7 HV 1 DK	7 HV 1 DK	7 HV 3 SS	3 HV	7 HV 2 SS 2 DK 1 LB	3 HV 1 LB 1 LO

- : No effects, fish observed as normal
DK: Dark – Darkened pigmentation.
HV: Hyperventilation – Rapid opercula movement.
SS: Surface swimming – Unable to swim down in water column.
LB: Lethargic at base – Slow moving / periods without movement, responds to stimuli. Mostly at base of tank.
LO: Loss of orientation – Loss of ability to maintain body position / unable to maintain upright swimming position.

Validity criteria fulfilled:

yes

Remarks:

The study met all of the validity requirements as outlined in the Study Plan and OECD Test Guideline.

Conclusions:

After 96 hours exposure to 4.27, 9.39, 20.7, 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L treatment rates, observed mortality was 0% in all test concentrations.Therefore, the 96-hour LC50 is >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. Sublethal effects were observed in the 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L treatment rates. The NOEC was 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L and LOEC was >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L.

Executive summary:

The aim of this study was to determine the effects of (2S)-5-oxopyrrolidine-2-carboxylic acid on freshwater fish species Oncorhynchus mykiss, (Rainbow Trout) under semi-static conditions. In this acute toxicity test, fish were exposed to the test item over a test period of 96 hours. The study met the requirements specified in the OECD Guidelines for Testing Chemicals No.203, Fish, Acute Toxicity Test (adopted 17 July 1992).
Based on the results of the range-finding bioassay, seven juvenile rainbow trout were exposed to nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. Renewal of the test solutions was performed at the 24 and 72 hour time points.
To verify actual exposure levels, analysis of (2S)-5-oxopyrrolidine-2-carboxylic acid was carried out on the freshly prepared test solutions at 0, 24 and 72 hours and the aged test solutions at 24, 72 and 96 hours. Results are reported as the nominal concentrations as the measured levels of (2S)-5-oxopyrrolidine-2-carboxylic acid at each sampling time point were maintained between 80% to 120% of the nominal throughout the bioassay.
Observations for sub-lethal effects and mortality in the control and test item groups were conducted at approximately 15 minutes and 1, 2, 3, 4, 5, 24, 48, 72 and 96 hours exposure.

The effect of (2S)-5-oxopyrrolidine-2-carboxylic acid on Oncorhynchus mykiss was assessed over 96 hours. After 96 hours exposure to (2S)-5-oxopyrrolidine-2-carboxylic acid, observed mortality in each treatment rate was 0 %. Therefore, the 96-hour LC50 is >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. The no observed effect concentration (NOEC) was 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L and lowest effect concentration (LOEC) was >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. Sub-lethal
effects were observed in the 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L treatment rates.

Description of key information

After 96 hours exposure to 4.27, 9.39, 20.7, 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L treatment rates, observed mortality was 0% in all test concentrations.Therefore, the 96-hour LC50 is >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L. Sub-lethal effects were observed in the 45.5 and 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L treatment rates. The NOEC was 100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L and LOEC was >100 mg (2S)-5-oxopyrrolidine-2-carboxylic acid/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L