N-(2-nitrophenyl)phosphoric triamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24/02/2022-14/10/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Substance type: urease inhibitor

Molecular mass (g mol-1) 216.13

Analysed purity: 99.6%

Appearance: yellow solid (source: SDS)

Analytical monitoring:

yes

Details on sampling:

The following specimen(s) were analysed in the analytical phase of the study:
Treatment Day 0, fresh Day 3, spent Day 5, fresh Day 7, spent
1 22ADR2-1 22ADR2-7 22ADR2-13 22ADR2-19
2 22ADR2-2 22ADR2-8 22ADR2-14 22ADR2-20
3 22ADR2-3 22ADR2-9 22ADR2-15 22ADR2-21
4 22ADR2-4 22ADR2-10 22ADR2-16 22ADR2-22
5 22ADR2-5 22ADR2-11 22ADR2-17 22ADR2-23
6 22ADR2-6 22ADR2-12 22ADR2-18 22ADR2-24

Treatment Day 12, fresh Day 14, spent Day 19, fresh Day 21, spent
1 22ADR2-25 22ADR2-31 22ADR2-37 22ADR2-43
2 22ADR2-26 22ADR2-32 22ADR2-38 22ADR2-44
3 22ADR2-27 22ADR2-33 22ADR2-39 22ADR2-45
4 22ADR2-28 22ADR2-34 22ADR2-40 22ADR2-46
5 22ADR2-29 22ADR2-35 22ADR2-41 22ADR2-47
6 22ADR2-30 22ADR2-36 22ADR2-42 22ADR2-48

Vehicle:

no

Details on test solutions:

The test item was soluble in the test medium. An aqueous stock solution was prepared by adding the test item to test medium, followed by treating with ultrasonic for 30 seconds and stirring for 10 minutes on a magnetic stirrer. The concentrations were prepared by serial dilutions of the stock solution A.

- the required volumes of the stock solution A were filled up to approximately 950 mL with test medium


- Appropriate amounts of algal suspension (dependent on the content of carbon C) were added and filled up to 1000 mL with test medium to ensure approximately 0.1 and 0.2 mg C/Daphnia/day was provided.

Test medium was prepared at least 1 day in advance and aerated for 2 hours before the 1st usage.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism: Daphnia magna STRAUS

Origin of the test organism: Landesanstalt für Umweltschutz Baden-Württemberg,
Griesbachstr. 1, 76185 Karlsruhe, Germany

Origin of the animals used in the test: holding and breeding occurred in the Test Facility under standardised laboratory conditions; test animals were derived from a healthy stock; no first brood progeny were used as test animals

Age of the Daphnia (at test start): < 24 hours and not first brood progeny

Breeding conditions: breeding was performed in glass beakers (50 mL) under the same conditions as in the test. Test animals were separated from the adults by means of a pipette.

Breeding medium: Elendt M4 medium
(according to the OECD Guideline 211)

Food: Alga Desmodesmus subspicatus purchased from MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany,

Feeding: 3 times per week, depending on age the Daphnia were fed with a defined suspension of algae:
1 day old: 0.5 mL/50 mL M 4
3 to 10 days old: 1.0 mL/50 mL M 4
> 10 days old: 1.5 mL/50 mL M 4

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The water hardness was measured to be 335 mg/L CaCO3.

Test temperature:

19.9 – 20.2 °C

pH:

7.811-8.101

Dissolved oxygen:

8.18-9.04 mg/L

Conductivity:

conductivity ≤ 10 µS/cm, measured: 0.05 µS/cm

Nominal and measured concentrations:

Test concentrations (nominal): 1.24, 3.71, 11.1, 33.3, 100.0 mg/L test item
Test concentrations
(time-weighted mean): 1.24, 3.73, 11.1, 33.3, 102.3 mg/L test item

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (approximately 60 mL)
- Aeration: no aeration of the vessels
- Renewal rate of test solution (frequency/flow rate): The test medium was renewed 3 times per week.
- Number of parent (Daphnia)/test vessel (= replicate): 1
- Number of daphnids/concentration: 10
- Number of test vessels/concentration: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:air-saturated deionized water (conductivity ≤ 10 µS/cm, measured: 0.05 µS/cm)
- Test medium: Elendt M4 medium
- Intervals of water quality measurement: Measurements of test medium parameters before test start and before each renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark, daily, light intensity: approximately 20 µE/m2s-1, equivalent to 1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Time and parameters

At test start, test end and at least once a week after test start:
- pH, dissolved oxygen content*
- water hardness*
- test item concentrations

Daily from the appearance of first brood:
- Assessment of offspring and dead parent animals (counting)
- Recording the time to first brood in each test vessel will be assessed

The temperature was measured continuously.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 102.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 102.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 102.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Development rate for reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Development rate for reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.73 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Development rate for reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.73 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

71.7 mg/L

95% CI:

>= 41.8 - <= 161.7

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.97 mg/L

95% CI:

>= 0.981 - <= 3.2

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.73 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

60.6 mg/L

95% CI:

>= 33.1 - <= 160.8

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

2.04 mg/L

95% CI:

>= 0.851 - <= 8.56

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Results for reproduction and mortality of Daphnia magna

Treatment group	mg/L N-(2-Nitrophenyl) phosphoric triamide
	control	1.24	3.71	11.1	33.3	100.0
	mg/L test item, TW mean
	control	1.24	3.73	11.1	33.3	102.3
21 days after application	Cumulative number of living offspring
Mean number per introduced parent	90.6	91.7	72.1	66.4	50.7	32.7
SD	8.36	7.06	10.95	9.85	6.93	13.16
CV (%)	9.2	7.7	15.2	14.8	13.7	40.2
Mean number per survived parent	90.6	91.7	72.1	66.4	50.7	36.3
SD	8.36	7.06	10.95	9.85	6.93	6.80
CV (%)	9.2	7.7	15.2	14.8	13.7	18.7
	Reduction (%) in cumulative number of offspring per introduced parent^1 relative to the control
	n.r.	-1.2	20.4 +	26.7 +	44.0 +	63.9 +
	Reduction (%) in cumulative number of offspring per survived parent^1 relative to the control
	n.r.	-1.2	20.4 +	26.7 +	44.0 +	59.9 +
	Mortality of parent daphnids (%)
	0.0	0.0	0.0	0.0	0.0	10.0
	Age of first reproduction (Days)^1
	10.8	11.1	11.8	11.1	12.8	13.1
	Development rate for reproduction
	0.093	0.090	0.085	0.090	0.078	0.077
	Inhibition of development rate for reproduction (%)^1 relative to the control
	n.r.	2.7	8.0 +	2.5 +	15.4 +	17.3 +
	Mean intrinsic rate (r) of population increase
	0.313	0.303	0.275	0.290	0.257	0.224
	Inhibition of intrinsic rate (r) of population increase (%)^1 relative to the control
	n.r.	3.1	12.1 +	7.2 +	18.0 +	28.5 +

Effects on reproduction of Daphnia magna: cumulative number of offspring and development rate

Effect concentration	mg/L N-(2-Nitrophenyl) phosphoric triamide
	Mean cumulative number of offspring per introduced parent 21 days after application	Mean cumulative number of offspring per survived parent 21 days after application	Development rate for reproduction
NOEC
test item, nominal	1.24	1.24	1.24
test item, TW mean	1.24	1.24	1.24
LOEC
test item, nominal	3.71	3.71	3.71
test item, TW mean	3.73	3.73	3.73
EC10	EC10	EC10	EC10
and 95 % confidence intervals
(lower – upper)
test item, nominal	2.04	1.97	6.48
	(0.830 – 3.59)	(0.966 – 3.22)	(n.d.)
test item, TW	2.04	1.97	6.50
	(0.851 – 3.56)	(0.981 – 3.20)	(n.d.)
EC20
and 95 % confidence intervals
(lower – upper)
test item, nominal	6.50	6.75	n.d.
	(3.71 – 10.5)	(4.31 – 10.1)
test item, TW mean	6.53	6.78	n.d.
	(3.77 – 10.5)	(4.36 – 10.0)
EC50
and 95 % confidence intervals
(lower – upper)
test item, nominal	59.9	70.9	n.d.
	(32.5 – 163.6)	(41.1 – 162.8)
test item, TW mean	60.6	71.7	n.d.
	(33.1 – 160.8)	(41.8 – 161.7)

 

Effects on reproduction of Daphnia magna: mortality of parents, age of first reproduction and intrinsic rate of population increase

Effect concentration	mg/L N-(2-Nitrophenyl) phosphoric triamide
	Mortality of parents	Age of first reproduction	Intrinsic rate of population increase*
NOEC
test item, nominal	≥ 100.0	1.24	1.24
test item, TW mean	≥ 102.3	1.24	1.24
LOEC
test item, nominal	> 100.0	3.71	3.71
test item, TW mean	> 102.3	3.73	3.73
EC10
and 95 % confidence intervals
(lower – upper)
test item, nominal	> 100.0	n.d.	9.35
			(3.98 – 22.0)
test item, TW mean	> 102.3	n.d.	9.28
			(3.93 – 21.9)
EC20
and 95 % confidence intervals
(lower – upper)
test item, nominal	n.d.	n.d.	44.8
			(17.3 – 120.7)
test item, TW mean	n.d.	n.d.	45.1
			(17.4 – 122.6)
EC50
and 95 % confidence intervals
(lower – upper)
test item, nominal	n.d.	n.d.	n.d.
test item, TW mean	n.d.	n.d.	n.d.

Calculations were conducted using unrounded values

TW mean - time-weighted mean

* The intrinsic rate is considered as a measure of population growth based on reproductive output and age-specific mortality. In steady state populations the intrinsic rate of population increase is zero, for growing populations it is positive and for shrinking populations it is negative.

n.d. - not determined due to mathematical reasons or inappropriate data

 

Validity criteria fulfilled:

yes

Conclusions:

A toxicity test was performed to assess the effects of the test item N-(2-Nitrophenyl) phosphoric triamide on the reproductive output of Daphnia magna during 21 days of exposure.

The measured test concentrations of the test item in test solutions ranged from 94 to 110% in fresh samples and from 65 to 117% in aged samples.
Based on the above, the results presented are based on the nominal concentrations for the test item as well as on the time-weighted mean concentrations.

The 21 day - NOEC, based on mean cumulative number of offspring per introduced parent, mean cumulative number of offspring per survived parent, development rate for reproduction, age of first reproduction and intrinsic rate of population increase is 1.24 mg/L test item (nominal concentration and TW mean).
The 21 day - NOEC, based on mortality of parents is ≥ 100.0 mg/L test item (equivalent to 102.3 mg/L test item, TW mean).

The EC10, EC20 and EC50 values were 2.04, 6.50 and 59.9 mg/L test item (equivalent to 2.04, 6.53 and
60.5 mg/L test item, TW mean) for mean cumulative number of offspring per introduced parent.

The EC10, EC20 and EC50 values were 1.97, 6.75 and 70.9 mg/L test item (equivalent to 1.97, 6.78 and
71.7 mg/L test item, TW mean) for mean cumulative number of offspring per survived parent.

The first offspring in control replicates was observed at day 10 after test start. No aborted brood or ephippia were observed in any treatment group.

Executive summary:

The primary objective of the test was to assess the effect of the test item on the reproductive output of Daphnia magna. The test duration was 21 days. At test end, the total number of living offspring produced was assessed. Data were analysed in order to determine the LOEC/NOEC values and to estimate concentrations that would cause a %-reduction in reproductive output (e.g. EC₁₀, EC₂₀ and EC₅₀ values at 21 days), if possible. Furthermore, parental mortality was determined. Replicates were excluded if mortality was caused by technical reasons (mishandling or accident).

The test was performed using a semi-static test design. The study was conducted in accordance with the recommendations of the OECD Test Guideline No. 211 (2012).

The measured test concentrations of the test item in test solutions ranged from 94 to 110% in fresh samples and from 65 to 117% in aged .

Based on the above, the results presented are based on the nominal concentrations for the test item as well as on the time-weighted mean concentrations.

The first offspring in control replicates was observed at day 10 after test start.

Based on the above, the results presented are based on the nominal concentrations for the test item as well as on the time-weighted mean concentrations.

The 21 day - NOEC, based on mortality of parents is ≥ 100.0 mg/L test item (equivalent to 102.3 mg/L test item, TW mean).

The EC₁₀, EC₂₀ and EC₅₀ values were 2.04, 6.50 and 59.9 mg/L test item (equivalent to 2.04, 6.53 and
60.5 mg/L test item, TW mean) for mean cumulative number of offspring per introduced parent.

The EC₁₀, EC₂₀ and EC₅₀ values were 1.97, 6.75 and 70.9 mg/L test item (equivalent to 1.97, 6.78 and 71.7 mg/L test item, TW mean) for mean cumulative number of offspring per survived parent.

Table 1: Effects on reproduction of Daphnia magna: cumulative number of offspring and development rate

Effect concentration	mg/L N-(2-Nitrophenyl) phosphoric triamide
	Mean cumulative number of offspring per introduced parent 21 days after application	Mean cumulative number of offspring per survived parent 21 days after application	Development rate for reproduction
NOEC
test item, nominal	1.24	1.24	1.24
test item, TW mean	1.24	1.24	1.24
LOEC
test item, nominal	3.71	3.71	3.71
test item, TW mean	3.73	3.73	3.73
EC10	EC10	EC10	EC10
and 95 % confidence intervals
(lower – upper)
test item, nominal	2.04	1.97	6.48
	(0.830 – 3.59)	(0.966 – 3.22)	(n.d.)
test item, TW mean	2.04	1.97	6.50
	(0.851 – 3.56)	(0.981 – 3.20)	(n.d.)
EC20
and 95 % confidence intervals
(lower – upper)
test item, nominal	6.50	6.75	n.d.
	(3.71 – 10.5)	(4.31 – 10.1)
test item, TW mean	6.53	6.78	n.d.
	(3.77 – 10.5)	(4.36 – 10.0)
EC50
and 95 % confidence intervals
(lower – upper)
test item, nominal	59.9	70.9	n.d.
	(32.5 – 163.6)	(41.1 – 162.8)
test item, TW mean	60.6	71.7	n.d.
	(33.1 – 160.8)	(41.8 – 161.7)

Table 2: Effects on reproduction of Daphnia magna: mortality of parents, age of first reproduction

and intrinsic rate of population increase

Effect concentration	mg/L N-(2-Nitrophenyl) phosphoric triamide
	Mortality of parents	Age of first reproduction	Intrinsic rate of population increase*
NOEC
test item, nominal	≥ 100.0	1.24	1.24
test item, TW mean	≥ 102.3	1.24	1.24
LOEC
test item, nominal	> 100.0	3.71	3.71
test item, TW mean	> 102.3	3.73	3.73
EC10
and 95 % confidence intervals
(lower – upper)
test item, nominal	> 100.0	n.d.	9.35
			(3.98 – 22.0)
test item, TW mean	> 102.3	n.d.	9.28
			(3.93 – 21.9)
EC20
and 95 % confidence intervals
(lower – upper)
test item, nominal	n.d.	n.d.	44.8
			(17.3 – 120.7)
test item, TW mean	n.d.	n.d.	45.1
			(17.4 – 122.6)
EC50
and 95 % confidence intervals
(lower – upper)
test item, nominal	n.d.	n.d.	n.d.
test item, TW mean	n.d.	n.d.	n.d.

 

 

 

 

Description of key information

A toxicity test was performed to assess the effects of the test item N-(2-Nitrophenyl) phosphoric triamide on the reproductive output of Daphnia magna during 21 days of exposure.

The measured test concentrations of the test item in test solutions ranged from 94 to 110% in fresh samples and from 65 to 117% in aged samples.
Based on the above, the results presented are based on the nominal concentrations for the test item as well as on the time-weighted mean concentrations.

The 21 day - NOEC, based on mean cumulative number of offspring per introduced parent, mean cumulative number of offspring per survived parent, development rate for reproduction, age of first reproduction and intrinsic rate of population increase is 1.24 mg/L test item (nominal concentration and TW mean).
The 21 day - NOEC, based on mortality of parents is ≥ 100.0 mg/L test item (equivalent to 102.3 mg/L test item, TW mean).

The EC10, EC20 and EC50 values were 2.04, 6.50 and 59.9 mg/L test item (equivalent to 2.04, 6.53 and
60.5 mg/L test item, TW mean) for mean cumulative number of offspring per introduced parent.

The EC10, EC20 and EC50 values were 1.97, 6.75 and 70.9 mg/L test item (equivalent to 1.97, 6.78 and
71.7 mg/L test item, TW mean) for mean cumulative number of offspring per survived parent.

The first offspring in control replicates was observed at day 10 after test start. No aborted brood or ephippia were observed in any treatment group.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

1.24 mg/L