Condensation products of phenol and salicylaldehyde

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:

23 September 2019 to 27 September 2019

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:

17 July 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1

Version / remarks:

The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13–Seisan-3986 on 10 October 2001.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sample storage conditions before analysis: 12 tubes of test solutions (3.2 mL aliquots) were received from the test facility. The samples were kept below - 15 °C before the analysis.
Duplicate samples were taken (2 x ~3.2 mL) in plastic tubes at the beginning and at the end of the test from the control and at the applied test concentration levels. After sampling, samples were frozen and kept approximately at - 20 °C at the test facility. One set of the samples was sent to the test site for analysis and one set was retained as a back-up at the test facility, if required for any confirmatory analyses (samples discarded after satisfactory results were obtained).
The test concentrations were analytically determined at the start and at the end of the experiment.

Vehicle:

no

Remarks:

Because the test material is a UVCB, test material solutions were prepared using a saturated solution method (water accommodated fraction (WAF)).

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Because the test material is a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials) and poorly soluble in water, test material solutions were prepared using a saturated solution method (water accommodated fraction (WAF)) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000).
- Saturated test material solutions (nominal loading rates of 6.25, 12.50, 25.00, 50.00 and 100.00 mg/L) were prepared individually by dispersing / dissolving the amount of test material into the test medium (aquarium water) two days before the start of the experiment. These solutions were shaken for about 24 hours at approximately 30 °C and then equilibrated for about 2 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solutions. The test material solutions were prepared just before introduction of the fish (start of the experiment).
- No correction for purity was applied.
- Untreated Control: The dilution water (circulated and filtered) was used without of addition of the test material.

SOLUTIONS, MIXTURES
Test media: aquarium water (according to OECD 203) was used as test media.
Diluent: Acetonitrile : Aquarium Water 35: 65 (V: V %) was used as a Diluent.
Stock solution: Approximately 10 mg test material was weighted with 0.01 mg precision into a 10 mL volumetric flask and filled up with diluent. The solution was mixed well by hand. The concentrations of the reference substances were approximately 1.0 mg/mL.
Working solution: The concentration of the working solution was 10 µg/mL.
Calibration solutions: The working solution was diluted in the 0.5 – 10 µg/mL range. The calibration solutions were injected in duplicates.

ANALYTICAL SAMPLE PREPARATION
The test material solutions were diluted into the dynamic range of the calibration in two steps with the diluent.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
Number of animals: Seven fish per test group.
Body length of animals: 2.5 – 3.3 cm.

ACCLIMATION
Acclimatisation: More than 12 days.
Animal health: Fish were bred in a well-known fish farm, under disease and parasite-controlled conditions. Fish were observed during the acclimatisation and test period, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- Amount: The fish were not fed during the test.

Test type:

static

Water media type:

other: Aquarium water (according to OECD 203) was used as the test medium.

Limit test:

no

Total exposure duration:

96 h

Hardness:

The hardness of the dilution water was determined as 176 mg/L (as CaCO3).

Test temperature:

The test temperature was between 21.0 °C and 23.8 °C.

pH:

The pH was in the range of 8.01 – 8.31.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 74 – 100 % of the air saturation value at the temperature used.

Nominal and measured concentrations:

The nominal concentrations of test material used in the main experiment were: 6.25, 12.50, 25.00, 50.00 and 100.00 mg/L nominal loading rates WAFs.
The corresponding measured geometric mean test material concentrations were: 4.70, 9.10, 21.66, 23.87 and 26.32 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: 5 L test material solution.
- No. of organisms per vessel: Seven fish per aquarium
- No. of vessels per concentration: One aquarium was used for the test groups.
- No. of vessels per control: One aquarium was used for the control group.
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test material solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: The water temperature, oxygen concentration and pH were measured at the beginning of experiment and 24-hours intervals thereafter in each test aquarium (where the fish were alive).

OTHER TEST CONDITIONS
- Photoperiod: The light-dark cycle during the test was 16 hours light and 8 hours darkness.

EFFECT PARAMETERS MEASURED
- The observations of fish were carried out in the following intervals: At 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub-lethal effect were determined in each observation period. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/L testing liquid).
- After the experiment, all survivor fish were humanely killed. Euthanasia for all animals was applied by an overdose of an aqueous solution of 250 mg/L tricaine methanesulphonate (MS 222; CAS No.: 886-86-2). This technique is considered to provide a humane euthanasia by direct depression of the brain and vital centres.

TEST CONCENTRATIONS
- The choice of the test concentrations was made on the basis of the results of the preliminary range-finding test.
- Range-finding study: A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Four fish were exposed to each concentration of the test material plus a control, for 96 hours under semi-static conditions. The frequency of the water renewal periods was 24 hours.
- During the formulation procedure the test material stock solution was prepared by similar method (with the exception of duration of equilibration which was 24 hours).
- Test concentrations: The range-finding study was conducted at 0, 0.1, 1.0, 10 and 100 mg/L nominal loading rate WAF using four animals at each concentration.
- Results used to determine the conditions for the definitive study: Because toxic effect was observed at the highest concentration level (100 mg/L WAF) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under static conditions.
The nominal concentrations of test material used in the main experiment were: 6.25, 12.50, 25.00, 50.00 and 100.00 mg/L nominal loading rates WAFs.
The biological results are based on both of the nominal loading rates WAFs and the measured geometric mean test material concentrations.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

12.5 other: mg/L nominal LR WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

25 other: mg/L nominal LR WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

9.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

21.66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

70.62 other: mg/L nominal LR WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 49.61 - 104.57 mg/L WAF

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

56.57 other: mg/L nominal LR WAF.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 41.52 - 106.60 mg/L WAF

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

52 other: mg/L nominal LR WAF.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 32.43 - 106.88 mg/L WAF

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

43.98 other: mg/L nominal LR WAF.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 20.52 - 60.95 mg/L WAF

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

100 other: mg/L nominal LR WAF.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

25.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 23.71 - 28.28 mg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

24.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 22.94 - 26.19 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 22.54 - 25.69 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

23.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % conf. limits: 21.74 - 24.75 mg/L

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

26.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDITY
The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST MATERIAL
The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs.
The test concentrations were analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test material concentrations were: 4.70; 9.10; 21.66; 23.87 and 26.32 mg/L. Exposure concentrations were calculated.
Biological results are related to both of the nominal loading rates WAFs and the measured geometric mean test material concentrations.

OBSERVATIONS
Behaviour of fish was found as normal in the control group and at the tested concentration levels of 6.25 and 12.50 mg/L nominal loading rates WAFs.
Localisation at the bottom of aquarium or close to the surface of the water, decreased activity and immobility was observed at the test concentrations of 25.00 and 50.00 mg/L nominal loading rates WAFs during the experiment.
At the highest test material group of 100.00 mg/L nominal loading rate WAF almost the same observations were recorded (localisation at the bottom of aquarium or close to the surface of the water and decreased activity) during the first 24 hours of the test, while all fish were dead 48 hours after treatment.

BODY WEIGHT
The body weight of 7 fish was weighed at the start of the test.
There was no considerable difference observed concerning body weights between the groups.

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LL50 / LC50 values were calculated by Probit analysis using SPSS PC+ software (based on the measured geometric mean concentrations).
The NOELR/ NOEC, LOELR/ LOEC and LL100/ LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Calculation of Exposure Concentrations

Nominal (mg/L Loading Rate WAF)	Measured Concentrations (mg/L)		Geometric Mean
	0 h/Start	96 h/End	mg/L
Control	n.d.	n.d.	-
6.25	4.636	4.773	4.70
12.50	8.849	9.353	9.10
25.00	21.286	22.050	21.66
50.00	23.235	24.513	23.87
100.00*	25.915	26.740	26.32

n.d.: Not detected.

* Concentration of the highest examined test group of 100.00 mg/L nominal loading rate WAF was analysed at the start of the experiment and 48 hours later when all animals were observed as dead.

Cumulative Mortality Data in the Definitive Test

Concentration		Cumulative Mortality [Number of Fish] (Initial Population = 7 Fish)
Nominal (mg/L Loading Rate WAF)	Measured (mg/L)	3h	6h	24h	48h	72h	96h
Control	-	0	0	0	0	0	0
6.25	4.70	0	0	0	0	0	0
12.50	9.10	0	0	0	0	0	0
25.00	21.66	0	0	0	0	0	0
50.00	23.87	0	0	1	2	3	5
100.00	26.32	0	0	6	7	-	-

 

Measured and Calculated Data of Bodyweight

Concentration		Measured Weight of 7 Fish (g)	Calculated Mean Weight of 1 Fish (g)	Loading of Testing Aquarium (g fish/L testing liquid)
Nominal (mg/L Loading Rate WAF)	Measured (mg/L)
Control	-	1.79	0.26	0.36
6.25	4.70	1.97	0.28	0.39
12.50	9.10	1.54	0.22	0.31
25.00	21.66	1.85	0.26	0.37
50.00	23.87	1.52	0.22	0.30
100.00	26.32	1.95	0.28	0.39

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96 h LL50 was 43.98 mg/L nominal loading rate WAF (95 % confidence limits of 20.53 - 60.95 mg/L WAF). The 96 h measured LC50 was 23.28 mg/L (95 % confidence limits of 21.74 - 24.75 mg/L).
The 96 hour No-Observed Effect Loading Rate (NOELR) was 12.50 mg/L nominal loading rate WAF and the 96 hour Lowest Observed Effect Loading Rate (LOELR) was 25.00 mg/L nominal loading rate WAF. The 96 hour No-Observed Effect Concentration (NOEC) was 9.10 mg/L (measured) and the 96 hour Lowest Observed Effect Concentration (LOEC) was 21.66 mg/L (measured).

Executive summary:

The acute toxicity of the test material was assessed with an acute fish toxicity test on Zebrafish (Brachydanio rerio) according to OECD Guideline 203 (Fish, Acute Toxicity Test), the EU Method C.1 (Acute Toxicity for Fish), the EPA Health Effects Test Guidelines OCSPP 850.1075 and JMAFF 2-7-1-1 (The guidelines related to the study reports for the registration application of pesticides) in compliance with GLP over an exposure period of 96 hours in a static system.

Because toxic effect was observed at the highest concentration level (100.00 mg/L WAF) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.50, 25.00, 50.00 and 100.00 mg/L nominal loading rates WAFs.

The test material concentrations were analytically determined at the start and at the end of the experiment. Concentration of the highest examined test group of 100.0 mg/L nominal loading rate WAF was analysed at the start of the experiment and 48 hours later when all animals were observed as dead.

The corresponding measured geometric mean test material concentrations were: 4.70; 9.10; 21.66; 23.87 and 26.32 mg/L.

The biological results are based on both of the nominal loading rates WAFs and the measured geometric mean test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litres of test solution.

The 24, 48, 72 and 96 hours LL50/LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOELR/NOEC and LOELR/LOEC and LL100/LC100 values were determined from the raw data.

All achievable validity criteria were met during this study. 

Under the conditions of the study the  96 h LL50 was 43.98 mg/L nominal loading rate WAF (95 % confidence limits of 20.53 - 60.95 mg/L WAF). The 96 h measured LC50 was 23.28 mg/L (95 % confidence limits of 21.74 - 24.75 mg/L).

The 96 hour No-Observed Effect Loading Rate (NOELR) was 12.50 mg/L nominal loading rate WAF and the 96 hour Lowest Observed Effect Loading Rate (LOELR) was 25.00 mg/L nominal loading rate WAF. The 96 hour No-Observed Effect Concentration (NOEC) was 9.10 mg/L (measured) and the 96 hour Lowest Observed Effect Concentration (LOEC) was 21.66 mg/L (measured).

Description of key information

Sipos (2020)

Under the conditions of the study the  96 h LL50 was 43.98 mg/L nominal loading rate WAF (95 % confidence limits of 20.53 - 60.95 mg/L WAF). The 96 h measured LC50 was 23.28 mg/L (95 % confidence limits of 21.74 - 24.75 mg/L).

The 96 hour No-Observed Effect Loading Rate (NOELR) was 12.50 mg/L nominal loading rate WAF and the 96 hour Lowest Observed Effect Loading Rate (LOELR) was 25.00 mg/L nominal loading rate WAF. The 96 hour No-Observed Effect Concentration (NOEC) was 9.10 mg/L (measured) and the 96 hour Lowest Observed Effect Concentration (LOEC) was 21.66 mg/L (measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

23.28 mg/L

Additional information

Sipos (2020)

The acute toxicity of the test material was assessed with an acute fish toxicity test on Zebrafish (Brachydanio rerio) according to OECD Guideline 203 (Fish, Acute Toxicity Test), the EU Method C.1 (Acute Toxicity for Fish), the EPA Health Effects Test Guidelines OCSPP 850.1075 and JMAFF 2-7-1-1 (The guidelines related to the study reports for the registration application of pesticides) in compliance with GLP over an exposure period of 96 hours in a static system.

Because toxic effect was observed at the highest concentration level (100.00 mg/L WAF) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.50, 25.00, 50.00 and 100.00 mg/L nominal loading rates WAFs.

The test material concentrations were analytically determined at the start and at the end of the experiment. Concentration of the highest examined test group of 100.0 mg/L nominal loading rate WAF was analysed at the start of the experiment and 48 hours later when all animals were observed as dead.

The corresponding measured geometric mean test material concentrations were: 4.70; 9.10; 21.66; 23.87 and 26.32 mg/L.

The biological results are based on both of the nominal loading rates WAFs and the measured geometric mean test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litres of test solution.

The 24, 48, 72 and 96 hours LL50/LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOELR/NOEC and LOELR/LOEC and LL100/LC100 values were determined from the raw data.

All achievable validity criteria were met during this study. 

Under the conditions of the study the  96 h LL50 was 43.98 mg/L nominal loading rate WAF (95 % confidence limits of 20.53 - 60.95 mg/L WAF). The 96 h measured LC50 was 23.28 mg/L (95 % confidence limits of 21.74 - 24.75 mg/L).

The 96 hour No-Observed Effect Loading Rate (NOELR) was 12.50 mg/L nominal loading rate WAF and the 96 hour Lowest Observed Effect Loading Rate (LOELR) was 25.00 mg/L nominal loading rate WAF. The 96 hour No-Observed Effect Concentration (NOEC) was 9.10 mg/L (measured) and the 96 hour Lowest Observed Effect Concentration (LOEC) was 21.66 mg/L (measured).