N-(2-nitrophenyl)phosphoric triamide

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:

25/04/2022 - 19/10/2022

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item name: N-(2-Nitrophenyl)-phosphoric triamide (2-NPT)
Substance name: N-(2-Nitrophenyl)-phosphoric triamide
CAS number: 874819-71-3
EC number: 447-690-9
Chemical formula: C6H9N4O3P
Molecular mass: 216.1 g/mol
Batch number: 210402
Purity: 99.6%

Analytical monitoring:

yes

Details on sampling:

Samples of selected stock and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Duplicate samples were kept separately as a reserve. Each sample was analysed independently.

Vehicle:

no

Details on test solutions:

Stock solutions (S1) of the test item in modified reconstituted water were prepared by weighing 2.50054–2.75041 g of the test item into 5 L of dilution medium, resulting in nominal concentrations of 500–550 mg test item/L (April 26, 2022 to May 25, 2022). This solution (5 L) was intensively stirred on a magnetic stirrer at 1100 rpm at room temperature for 15 minutes interrupted by treating the solution in an ultrasonic bath (in total approx. 2 minute) to ensure a homogeneous distribution of the test item in the test medium.Thereafter the stock solution (S1), containing the pre-dissolved test item, was combined with the total amount of test medium used to prepare the final stock solution (S2). Therefore, the stock solution (S1) was transferred in a stainless-steel tank. The measuring flask containing the stock solution S1 was rinsed once with modified reconstituted water, filled with test medium up to calibration mark, and added to the storage tank of the final stock solution (S2). The remaining amount of modified reconstituted water was added to the stock solution tank by using a 10 L measuring flask ether prior to or directly after addition of the stock solution (S1) and the rinsing solutions, so that in total 100–110 L of modified reconstituted water was provided in the stainless-steel tank.
This final stock solution was stirred for 1–2 minutes at ambient temperature in the dark using a stainless-steel propeller stirrer.
The test solutions were prepared by a flow-through device. The stock solution S2 and the dilution medium (test medium) were delivered by the flow-through system to mixing vessels. The final test solutions were delivered directly from the mixing vessels to the designated test vessels using Teflon tubing and a multi-channel peristaltic pump assembled with Tygone® tubes, set to deliver the desired volume. During dosing into the flow-through system (between renewals), the control and the stock solution were stored at room temperature in the dark. During storage, the tanks were closed with a lid to reduce air-exchange and evaporation.
The flow rates (volume delivered per stroke) were measured volumetrically at least three times per week. The volume delivered/measured was divided by the number of strokes. The variation of the nominal stroke-volume was calculated by dividing the measured flow [mL/stroke] by the nominal volume per stroke of the designated pump expressed in percentage of nominal values.
The actual measured flow rates for all treatments were within ±10% of the mean flow rates during the exposure period, except for the treatment level C1 (1.00 mg/L) and C2 (1.77 mg/L) the flow rate was slightly higher at 2 measuring intervals with a maximum of 5.51-times turn-over rate.The test vessels containing 800 mL of the test solution were placed into a temperature-controlled water bath at test temperature for temperature adaptation prior to test start.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Danio rerio Hamilton-Buchanan 1822 (Zebrafish).

Origin: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since Jan/May 2021.

Holding conditions and fertilisation of test organisms: In the culture tanks, the fish were kept in reconstituted water diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ± 2‰). Fish were held in glass aquaria at 26 ± 2 °C with a 16 h/8 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, U.S.A.) and TetraMin (Tetra Werke, Melle, Germany).

Test type:

flow-through

Water media type:

other: Modified reconstituted water

Limit test:

no

Total exposure duration:

35 d

Hardness:

164-179 mg CaCO3/L

Test temperature:

25.5 - 26.9 °C

pH:

7.2 - 7.9

Dissolved oxygen:

5.6 - 8.7

Nominal and measured concentrations:

Nominal: 1.00, 1.77, 3.16, 5.62 and 10.0 mg test item/L plus a control
The measured concentration of the test item within the test solutions (treatment levels) showed stable concentration throughout the exposure period and no significant decline of the test item concentration compared to the time of the exposure period. The recoveries calculated based on the nominal concentrations in mg test item/L ranged between 71.5% and 106.9%, except in one sample of the highest treatment level (C5) with a recovery of 52.7%; this sample was excluded from further evaluation, see explanation below table above. Since, some recoveries were determined to be below 80% of nominal it was decided to calculate the mean measured concentration for each treatment level.

To calculate the mean measured concentrations of N-(2-Nitrophenyl)-phosphoric triamide (2-NPT) at each treatment level, the geometric mean concentration of each concentration level C1 to C5 (1.00 to 10.0 mg test item/L) was calculated. The resulting mean measured concentrations were 0.869, 1.54, 2.79, 5.41 and 9.42 mg test item/L for C1 to C5, respectively, equivalent to mean recoveries of 86.9, 87.0, 88.3, 96.3 and 94.2% for C1 to C5, respectively.

The biological data were reported based on nominal test item concentrations in mg test item/L, and based on mean measured test item concentrations in mg test item/L.

Key result

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Number of healthy fish

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Number of healthy fish

Key result

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success

Hatching Success

Nominal concentration [mg test item/L]	Introduced eggs [n]	Hatched fish [n]	Unhatched fish [n]	Hatching success [%]
0 (Control)	80	79	1	98.8
1.00 (C1)	80	79	1	98.8
1.77 (C2)	80	78	2	97.5
3.16 (C3)	80	77	3	96.3
5.62 (C4)	80	78	2	97.5
10.0 (C5)	80	75	5	93.8

Post-Hatch Success (Survival) of the Fish

Nominal concentration [mg test item/L]	Hatched fish larvae [n]	Surviving fish larvae [n]	Dead fish larvae [n]	Post-hatch mortality [% of hatched eggs]	Post-hatch success (survival) [% of hatched eggs]
0 (Control)	79	69	10	12.7	87.3
1.00 (C1)	79	72	7	8.9	91.1
1.77 (C2)	78	74	4	5.1	94.9
3.16 (C3)	77	69	8	10.4	89.6
5.62 (C4)	78	72	6	7.7	92.3
10.0 (C5)	75	69	6	8.0	92.0

Wet Weight of the Surviving Fish at Test End

Nominal concentration [mg test item/L]	Mean [mg]	Standard deviation [mg]	Difference compared to control [%]
0 (Control)	40.065	2.9683	–
1.00 (C1)	36.035	2.3599	10.1
1.77 (C2)	38.985	2.0642	2.7
3.16 (C3)	38.295	2.2744	4.4
5.62 (C4)	37.530	2.4388	6.3
10.0 (C5)	39.712	2.7009	0.9

Dry Weight of the Surviving Fish at Test End

Nominal concentration [mg test item/L]	Mean [mg]	Standard deviation [mg]	Difference compared to control [%]
0 (Control)	11.13	0.865	–
1.00 (C1)	10.01	0.847	10.1
1.77 (C2)	10.82	0.690	2.8
3.16 (C3)	10.70	0.840	3.9
5.62 (C4)	10.12	0.511	9.1
10.0 (C5)	10.90	1.000	2.1

Length of the Fish at Test End

Nominal concentration [mg test item/L]	Mean [mm]	Standard deviation [mm]	Reduction compared to control [%]
0 (Control)	17.36	0.362	–
1.00 (C1)	16.91	0.289	2.6
1.77 (C2)	17.25	0.317	0.6
3.16 (C3)	17.23	0.399	0.7
5.62 (C4)	17.02	0.452	1.9
10.0 (C5)	17.31	0.363	0.3

Summary of Biological Results

Nominal concentration [mg test item/L]	Introduced eggs [n]	Hatched fish [n]	Survived fish [n]	Deformed fish/ different behaviour [n]	Healthy fish [n]	Wet weight of fish [mg]	Dry weight of fish [mg]	Length of fish [mm]
0 (Control)	80	79	69	0	69	40.07	11.13	17.36
1.00 (C1)	80	79	72	0	72	36.04	10.01	16.91
1.77 (C2)	80	78	74	0	74	38.99	10.82	17.25
3.16 (C3)	80	77	69	0	69	38.30	10.70	17.23
5.62 (C4)	80	78	72	0	72	37.53	10.12	17.02
10.0 (C5)	80	75	69	0	69	39.71	10.90	17.31

 

 

Validity criteria fulfilled:

yes

Conclusions:

An Early-life Stage Toxicity Test (Semi-Static System) of BDP to Zebra-fish was conducted in accordance with OECD test guideline 210 with GLP compliance. The study was conducted under flow-through conditions with the nominal concentration of 1.00, 1.77, 3.16, 5.62 and 10.0 mg test item/L and control for a period of 35 days after post-hatch. The mortality/survival at embryo, larval and juvenile stages were observed and recorded once a day during exposure. At the end of the exposure, the total dry and wet weight of all surviving fish was determined and the length of surviving fish was measured. Compared with control group, the NOEC for the parameters hatching success, post-hatch success (Survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish and dry weight of surviving fish was estimated to be greater than 10 mg/L.

Executive summary:

An Early-life Stage Toxicity Test (Semi-Static System) of BDP to Zebra-fish was conducted in accordance with OECD test guideline 210 with GLP compliance. The study was conducted under flow-through conditions with the nominal concentration of 1.00, 1.77, 3.16, 5.62 and 10.0 mg test item/L and control for a period of 35 days after post-hatch. The mortality/survival at embryo, larval and juvenile stages were observed and recorded once a day during exposure. At the end of the exposure, the total dry and wet weight of all surviving fish was determined and the length of surviving fish was measured.

Under the experimental conditions of this study, no concentration-response relationship was observed for the parameter hatching success, post-hatch success (survival), length and wet weight of the surviving fish. The biological endpoints (NOEC/LOEC, EC₅₀) were reported based on nominal concentrations in the test solutions given in mg test item/L.

	Endpoints (nominal) [mg test item/L]
Parameter	EC50	NOEC	LOEC
Hatching success	n.d.	≥10.0	>10.0
Post-hatch success (Survival)	n.d.	≥10.0	>10.0
Number of healthy fish^1	n.d.	≥10.0	>10.0
Length of the surviving fish	n.d.	≥10.0	>10.0
Wet weight of the surviving fish	n.d.	≥10.0	>10.0
Dry weight of surviving fish	n.d.	≥10.0	>10.0

Analytical Findings

To determine the actual test item concentrations, samples were taken from the stock and test solutions for analytical measurement. Samples were measured in test solutions at regular intervals throughout the exposure period.

The measured concentration of the test item within the test solutions (treatment levels) showed stable concentration throughout the exposure period and no significant decline of the test item concentration compared to the time of the exposure period. The recoveries calculated based on the nominal concentrations in mg test item/L ranged between 71.5% and 106.9%, except in one sample of the highest treatment level (C5) with a recovery of 52.%. Since, some recoveries were determined to be below 80% of nominal it was decided to calculate the mean measured concentration for each treatment level.

To calculate the mean measured concentrations of N-(2-Nitrophenyl)-phosphoric triamide (2-NPT) at each treatment level, the geometric mean concentration of each concentration level C1 to C5 (1.00 to 10.0 mg test item/L) was calculated. The resulting mean measured concentrations were 0.869, 1.54, 2.79, 5.41 and 9.42 mg test item/L for C1 to C5, respectively, equivalent to mean recoveries of 86.9, 87.0, 88.3, 96.3 and 94.2% for C1 to C5, respectively.

 

 

Description of key information

An Early-life Stage Toxicity Test (Semi-Static System) of BDP to Zebra-fish was conducted in accordance with OECD test guideline 210 with GLP compliance. The study was conducted under flow-through conditions with the nominal concentration of 1.00, 1.77, 3.16, 5.62 and 10.0 mg test item/L  and control for a period of 35 days after post-hatch. The mortality/survival at embryo, larval and juvenile stages were observed and recorded once a day during exposure. At the end of the exposure, the total dry and wet weight of all surviving fish was determined and the length of surviving fish was measured. Compared with control group, the NOEC for the parameters hatching success, post-hatch success (Survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish and dry weight of surviving fish was estimated to be greater than 10 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 10 mg/L