2-(acetylamino)-N-benzyl-3-methoxypropanamide

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:

08 Jan 2018 - 23 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Completely soluble in test medium up to 100 mg/L.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the control.
- Sampling method: 1.8 mL at t=0 h, t=24 h and t=96 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition method: A 100 mg/L stock solution of test item in test medium was prepared by applying 15-20 minutes of ultrasonic waves to accelerate dissolution of the test item. This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Strain: Cyprinus carpio Linnaeus 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 3.4 ± 0.4 cm
- Weight at study initiation: 0.57 ± 0.25 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water.

HOLDING
- Acclimation period: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis)
- Feeding: Daily with pelleted fish food (Essence 300-500 µm, Coppens International BV, the Netherlands)
- Water quality parameters: Within the optimum limits for the respective fish species.
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

180 mg CaCO3/L

Test temperature:

21 - 22°C

pH:

7.5 - 7.9

Dissolved oxygen:

Day 0: 9.0-9.4 mg/L
Day 1: 7.4 mg/L
Day 2: 6.6-6.8 mg/L
Day 3: 6.1-6.2 mg/L
Day 4: 5.9-6.1 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L (combined range-finding/limit test)
Measured concentrations: only the 100 mg/L sample was analysed. At t=0, 24 and 96 h the measured concentrations were 104, 105 and 104 mg/L, respectively. Effect parameters were expressed based on analytically confirmed nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessels:
* Control and highest concentration: 10 litres, all-glass, containing 9 litres of test solution.
* Intermediate concentrations: 6.5 litres, all-glass, containing 4 litres of test solution.
- Aeration: no aeration
- Feeding: No feeding from 24 hours prior to the test and during the total test period.
- Renewal of test solution: no.
- No. of organisms per vessel: 7 (control and highest concentration), 3 (intermediate concentrations)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading: 0.44 g fish/L (control and highest concentration), 0.43 g fish/L (intermediate concentrations)
- Introduction of fish: Within 7 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 1.0°C.
- Terminal procedure: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH and temperature: Daily in the control and highest test concentration, beginning at the start of the test (day 0). pH and temperature was also measured in all other concentration at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily.

EFFECT PARAMETERS MEASURED
- Mortality and other effects: at 3¾, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP, performed February 2017)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Nominal test concentration confirmed by analysis.

Details on results:

- No mortality or other symptoms were observed throughout the test.
- Faeces were observed in the control, 1.0 and 100 mg/L at 24 hour of exposure.
- All water quality parameters remained within the limits as prescribed by the Study Plan.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 96h-LC50: 0.32 mg/L (95% confidence interval 0.22-0.46 mg/L)
- The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed at the test facility approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Sublethal observations / clinical signs:

Table 1: Test Sample concentrations

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	0.0053 #	n.a.
	100	104	104
24	0	0.0081 #	n.a.	n.a.
	100	105	105	102
96	0	0.0058 #	n.a.	n.a.
	100	104	104	101

# Estimated value, calculated using the mean response factor of the lowest calibration solution. A similar response was observed in the analytical blank.

n.a. Not applicable.

Table 2: Incidence of Mortality and Total Mortality

Nominal conc. (mg/L)	Initial Number of fish	Cumulativemortality					Total Mortality (%)
		3¾h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
0.10	3	0	0	0	0	0	0
1.0	3	0	0	0	0	0	0
10	3	0	0	0	0	0	0
100	7	0	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 96h-LC50 for Cyprinus carpio exposed to the test item exceeded the analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the substance to Cyprinus carpio was investigated. In a combined limit/range-finding test, performed under static conditions, carp were exposed for 96 hours to an untreated control and nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L (control and 100 mg/L – 7 carp per treatment group; 0.10, 1.0 and 10 mg/L – 3 carp per treatment group).

As the substance was completely soluble in test medium up to 100 mg/L, a stock solution of the substance at 100 mg/L was prepared (applying a 15-20 minute period of ultrasonication to accelerate dissolution of the test item). This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Samples for analytical confirmation of actual exposure concentrations were taken at t = 0, 24 and 96 hours from all test levels, but only the control and the highest concentration samples from these sampling times were analysed. The measured concentration in the highest test concentration was found to be at the level of the nominal concentration throughout the test (i.e. 104-105% of nominal). Since the test concentration remained stable during the test (101-102% of initial) the effect parameter was reported in terms of the analytically confirmed nominal concentration.

No mortality was observed in any of the test groups. Therefore, the 96h-LC50 of the substance to Cyprinus carpio is concluded to be > 100 mg/L.

The study met the OECD validity criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L