Nonanamide, N,N-dimethyl-

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:

06.2017 - 08.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes

Vehicle:

no

Details on test solutions:

Good quality tap water which had been dechlorinated for at least 24 hours was used. The total hardness of the dilution water was 121.6 mg (CaCO3)/L and pH was 8.08 at room temperature. Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The latest measuring result is showed

In the test, test solutions of the test substance were prepared by directly dissolving appropriate amount of test substance in dilution medium and then facilitating its dispersion by stirring for 30 min. The test substance was completely dissolved in water and the test solution was clear and transparent.
The details of the test solutions preparation were as follows:
Test Type Nominal Concentration (mg/L) Test Substance added
(g) Dilution Medium Volume (L)
Range-finding Test Blank control 0 3
1.00 0.0031 3
10.0 0.0307 3
100 0.3059 3
Definitive Test Blank control 0 3
10.0 0.0307 3
20.0 0.0617 3
40.0 0.1227 3
60.0 0.1852 3
80.0 0.2461 3
100 0.3052 3

Test organisms (species):

other: Gobiocypris rrus (Rare minnow)

Details on test organisms:

The Rare minnow, Gobiocypris rarus was used in the test on the basis of such important practical criteria as, for example, their ready availability throughout the year, ease of maintenance, convenience for testing and any relevant economic, biological or ecological factors. The test species of Rare minnow, Gobiocypris rarus (Batch No.: F20170410G), were bred by our own lab. A population of a single stock was used in the test.
(1) Holding of fish
Fish to be used in the definitive test were held for 7 days in water of the quality and temperature to be used in the test.
A photoperiod of 12 hours light, provided by overhead fluorescent tubes, and 12 hours dark was maintained. The oxygen concentration was 88% ~ 94% of the air saturation value. And the temperature was controlled between 22.8°C to 23.2°C, and pH value was between 7.65 to 7.77.
The fish was fed daily during the holding period on proprietary fish food. They were held without food for approximately 24 hours before being placed into the test vessels. The ingredients of the fish food are given as follows:
Crude Protein > 30.0%
Crude Fat > 2.0%
Crude Fibre < 3.0%
Crude Ash < 12%
Moisture < 10.0%
Characteristics of the fish food are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The latest measuring result is showed in Table 11.
(2) Criteria of fish selection
During the holding period the tanks was inspected daily and any debris or unhealthy or dead fish removed.
After the 48 hour settling-in period, no mortality was observed in the following 7 days. So the batch of fish (Batch No.: F20170410G) was accepted.
The average length and wet-weight of the fish used to the test was 2.43 cm and 0.222 g, and the relative standard deviation (RSD) was 4.69% and6.13%, respectively

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

148 - 161 mg/L CaC=3

Test temperature:

22.9 - 23.1 °C

pH:

7.63 - 7.84

Dissolved oxygen:

85 - 97 Dissolved oxygen (%)

Nominal and measured concentrations:

0-10-20-40-60-80-100 mg/l nominal

Details on test conditions:

During the test, all kinds of abnormal responses of the fish observed were recorded, such as mortality, inactivity, abnormal swimming pattern, other abnormal behaviour, etc. Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction.

Reference substance (positive control):

yes

Remarks:

Reference substance: Potassium dichromate, K2Cr2O7, CAS: 7778-50-9; purity: 99.8%; Lot number: LVC0O52; Beijing J&K Technology Co. Ltd. The test with the reference substance is performed at least once each batch of fish as a means of assuring that the lab

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

25.9 mg/L

95% CI:

19.9 - 33.5

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

Toxicity of Potassium Dichromate to Gobiocypris rarus
Nominal Concentration (mg/L) Initial Number
of Fish The Number of the Dead Fish
6 h 12 h 18 h 24 h
0 10 0 0 0 0
100 10 0 0 0 0
200 10 0 1 2 2
300 10 1 3 4 3
400 10 2 3 4 6
500 10 4 5 5 10
LC50 (mg/L) >500 500 500 310
95% confidence limit (mg/L) - - - 256-374

Sublethal observations / clinical signs:

Results

6.1 Analytical Method for Determination of the test substance in Water

(1) Specificity

Under the UPLC-PDA condition, retention time of test substance is 1.65 min. The chromatographic peak emerged for the test substance sample (Figure 2) and there was no chromatographic peak emerged for the blank mixture (acetonitrile). So the UPLC-PDA method was specific for N,N-Dimethylnonanamide.

(2) Calibration curve

A series of standard solutions with concentration at 1.00, 2.00, 5.00, 10.0, 20.0, 50.0 mg/L were measured under the UPLC-PDA conditions mentioned above. Based on the test result, a linear regression equation was obtained between the concentration and the UPLC-PDA response: A =101002c+12931 with good linearity of r² = 0.9982, where A represents peak areas (μV*s), and c is the concentration of the test substance (mg/L) (see Figure 1). The results showed that linearity for concentration range of 1.00 mg/L to 50.0 mg/L is good.

(3) Precision

Under the above condition, 10.0 mg/L solution of the test substance was analyzed for 6 times, the results were shown in Table 2. The relative standard deviation of the peak area was 1.31%.

(4) Recovery test

Recovery samples with concentrations of 10.0 mg/L were prepared by adding 0.401 mL standard solution the test substance I (499 mg/L) to a total volume of 20.0 mL test water with three replications. The recovery samples were filtered with 0.22 μm millipore membrane (discarded initial 2 mL), the filtrates were analyzed by UPLC-PDA.

Recovery samples with concentrations of 40.0 mg/L were prepared by adding 1.60 mL standard solution the test substance I (499 mg/L) to a total volume of 20.0 mL test water with three replications. The recovery samples were filtered with 0.22 μm millipore membrane (discarded initial 2 mL), the filtrates were analyzed by UPLC-PDA.

Measurements obtained from the recovery test were shown in Table 3. The recovery rate was 99.8% for the concentration of 10.0 mg/L and 90.0% for the concentration of 40.0 mg/L, respectively. The relative standard deviation was 0.347% and 0.556%, respectively.

(5) Limit of Detection (LOD) and Limit of Quantification (LOQ)

If the calculation is based on S/N≥3, the LOD is 0.2 mg/L. If the calculation is based on S/N≥10, the LOQ is about 0.6 mg/L.

6.2 Analysis of the test substance in Test Solutions

The analyzed results for the test samples from the definitive test are given in Table 4. Figure 4 and Figure 5 are the UPLC-PDA chromatogram of the control sample and test solution. The measured concentrations of 10.0, 20.0, 40.0, 60.0, 80.0 and 100 mg/L were 9.98, 18.4, 37.8, 61.1, 83.0 and 98.9 mg/L, respectively. The analytical results showed that the concentration of the test substance is consistent in the test medium throughout the 96-hour test period (deviation within 20%). Thus a semi-static conditions (24 h renewal) procedure was reasonable.

6.3 Test Condition

During the definitive test, the pH, dissolved oxygen concentration, total hardness and temperature of the control and treatment groups were showed in Table 5.

During the whole test period, the pH values of the control mediums and test mediums were between 7.63 and 7.84, the Dissolved Oxygen (DO) values varied from 85% ~ 97% of the air saturation, the temperature of the test mediums was maintained in the range of 22.9ºC to 23.1ºC, and the total hardness was in the range of 148 mg (CaCO3)/L to 161 mg (CaCO3)/L.

6.4 Mortality and Effects

Table 7 and Table 8 showed the mortality data during the range-finding test and definitive test respectively. Table 9 showed the summary of the visual observations (for behaviour or abnormalities) during the definitive test. During the test period, all fish in the control were alive and appeared normal. No fish die in 10.0 mg/L (mean measured concentration 9.98 mg/L) treatment. Effects (Fish lying on side or back) occurred at concentrations of 20.0 mg/L (mean measured concentration 18.4 mg/L) and above. All Fish were dead after 96 h-exposure at concentration of 60.0 mg/L (mean measured concentration 61.1 mg/L), 80.0 mg/L (mean measured concentration 83.0 mg/L) and 100 mg/L (mean measured concentration 98.9 mg/L).  

Conclusions:

The results showed that under valid semi-static conditions (24 h renewal), the 96 h-LC50 of the test substance to fish (Rare minnow, Gobiocypris rarus) was 25.9 mg/L, with 95% confidence limit of 19.9 mg/L ~ 33.5 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) was 10.0 mg/L (mean measured concentration 9.98 mg/L). The minimum concentration causing 100% mortality (96 h-LC100) was 60.0 mg/L (mean measured concentration 61.1 mg/L), i.e.:
96 h-LC50 = 25.9 mg/L, 95% CI 19.9 ~ 33.5 mg/L (based on measured concentration);
96 h-LC0 = 10.0 mg/L (mean measured concentration 9.98 mg/L);
96 h-LC100 = 60.0 mg/L (mean measured concentration 61.1 mg/L).

Executive summary:

Under semi-static conditions (24 h renewal), the acute toxicity of test substance (N,N-Dimethylnonanamide) to Rare minnow (Gobiocypris rarus) was conducted according to: “The guidelines for the testing of chemicals” (HJ/T 153-2004); and “The Guidelines for the Testing of Chemicals-Effects on Biotic Systems” (the 2^nd edition. Beijing: China Environment Press. 2013); Chemicals-Fish acute toxicity test” (GB/T 27861-2011); Chemicals- Rare minnow (Gobiocypris rarus) acute toxicity test (GB/T 29763-2013); with reference to Procedure 203 of the “Guidelines for Testing of Chemicals” of the OECD: “Fish, Acute Toxicity Test” (1992).

A range-finding test and then a definitive test was performed respectively. Nominal concentrations of 1.0, 10.0 and 100 mg/L were used in the range-finding test. While the nominal concentrations of 10.0, 20.0, 40.0, 60.0, 80.0 and 100 mg/L were used in the definitive test. Water samples taken from the blank control and the treatments in the definitive test were analysed. Concentrations of the test substance were 9.98, 18.4, 37.8, 61.1, 83.0 and 98.9 mg/L, respectively. The analytical results showed that the concentration of the test substance was consistent in the test medium throughout the 96-hour test period (deviation within 20%). Thus a semi-static conditions (24 h renewal) procedure was reasonable.

In the range-finding test, 5 fishes per treatment and no replicates were used. And 10 fishes per treatment of no replicates were used in the definitive test. The test fishes were exposed for 96 hours to the test solution.

During the test period, the pH values of the control mediums and test mediums were between 7.63 and 7.84, the Dissolved Oxygen (DO) values varied from 85% ~ 97% of the air saturation at the test temperature, the temperature of the test mediums were maintained in the range of 22.9ºC to 23.1ºC, and the total hardness was in the range of 148 mg (CaCO3)/L to 161 mg (CaCO3)/L. All fishes in the control group were normal. With the same conditions, K₂Cr₂O₇ was used as the positive control substance, and the resulting 24 h-LC₅₀ was 310 mg/L. So the study met the acceptability criteria prescribed by the protocol (The mortality of control ≤ 10%; pH: 6.0 ~ 8.5; dissolved oxygen concentration: >60% of the air saturation value; total hardness: 10 ~ 250 mg (CaCO₃)/L; temperature: (23±2)ºC; 24 h-LC₅₀ of K₂Cr₂O₇ in the range of 200 mg/L to 400 mg/L). Therefore the test was considered valid.

During the test period, all fish in the control were alive and appeared normal. No fish die in 10.0 mg/L (mean measured concentration 9.98 mg/L) treatment. Effects (Fish lying on side or back) occurred at concentrations of 20.0 mg/L (mean measured concentration 18.4 mg/L) and above. All Fish were dead after 96 h-exposure at concentration of 60.0 mg/L (mean measured concentration 61.1 mg/L), 80.0 mg/L (mean measured concentration 83.0 mg/L) and 100 mg/L (mean measured concentration 98.9 mg/L). The results showed that under valid semi-static conditions (24 h renewal), the 96 h-LC₅₀ of the test substance to fish (Rare minnow, Gobiocypris rarus) was 25.9 mg/L, with 95% confidence limit of 19.9 mg/L ~ 33.5 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC₀) was 10.0 mg/L (mean measured concentration 9.98 mg/L). The minimum concentration causing 100% mortality (96 h-LC₁₀₀) was 60.0 mg/L (mean measured concentration 61.1 mg/L), i.e.:

96 h-LC₅₀ = 25.9 mg/L, 95% CI 19.9 ~ 33.5 mg/L (based on measured concentration);

96 h-LC₀ = 10.0 mg/L (mean measured concentration 9.98 mg/L);

96 h-LC₁₀₀ = 60.0 mg/L (mean measured concentration 61.1 mg/L).

Description of key information

The results showed that under valid semi-static conditions (24 h renewal), the 96 h-LC50 of the test substance to fish (Rare minnow, Gobiocypris rarus) was 25.9 mg/L, with 95% confidence limit of 19.9 mg/L ~ 33.5 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) was 10.0 mg/L (mean measured concentration 9.98 mg/L). The minimum concentration causing 100% mortality (96 h-LC100) was 60.0 mg/L (mean measured concentration 61.1 mg/L), i.e.:
96 h-LC50 = 25.9 mg/L, 95% CI 19.9 ~ 33.5 mg/L (based on measured concentration);
96 h-LC0 = 10.0 mg/L (mean measured concentration 9.98 mg/L);
96 h-LC100 = 60.0 mg/L (mean measured concentration 61.1 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

25.9 mg/L