1-nitroguanidine

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:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Waller, Lazochak. 1986. Proposed new standard guide for conducting three brood, renewal toxicity tests with C. dubia. Draft No. 3 American Society for Testing and Materials.

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier U.S Naval Ordnance Station
Lot No 985-1
Essay 99.998%

Analytical monitoring:

yes

Details on sampling:

- Water quality and samples for HPLC measurements were taken daily in both replicates of all treatments when the test solutions were renewed
- Sample storage conditions before analysis: in the cases samples could not be analysed immediately following filtration, the filtered samples were stored at 4 °C in amber glass vials fitted with Teflon-lined caps and analyzed within 24 h from the time the samples were originally taken from the test aquaria.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Saturated stock solutions were prepared by dissolving appropriate amounts of matrial in aerated diluent water
- Stock solutions were stirred in the dark for 24h at room temperature
- Occasionally the test material was heated to ~30 °C
- All stock solutions were filtered before use to remove particles > 0.45 µm as well as excess reagent crystals in saturated solutions
- All stock solutions were prepared in amber glass containers
- The highest toxicant concentration tested was the solubility limit of the test material in JHU/APL (John Hopkins University Applied Physics Laboratory) well water at the test temperature
- Controls: test solution without nitroguanidine


PREPARATION AND APPLICATION OF PHOTOLYZED TEST SOLUTION
- Photolyzed nitroguanidine was prepared by exposing the compounds in JHU/APL well water to sunlight (38° 51' N latitude; 76° 31' W longitude) until the parent compound reached non-detectable levels as determined bx periodic HPLC analysis
- A 5L stock solution of 100 mg/l nitroguanidine (measured concentration) was photolyzed in direct sunlight in mid september for a total of 80 h until nitroguanidine could not be detected
- The stock solution of nitroguanidine, which was photolyzed in rectangular glass containers (40 x 58 x 82 cm) with no tops, were covered each evening with black plastic so that the actual number of hours of exposure to sunlight could be calculated
- Light intensity was not estimated by the use of a chemical actinometer

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Crustacea
- Source: Center for Lake Superior Environmental Studies, University of Wisconsin - Superior
- Holding Conditions:
- Temperture: 25 +/- 1 °C
- 600 ml glass beakers with 400 ml JHU/APL well water amended with selenium (2 µg Se/l as NaSeO3)
- Feeding: * Food type: mixture of Cerophyl and the green alga, S. capricornutum
* Amount: 120 µg Cerophyl/ml and 6.7x10°s S. capricornutum cells/ml
- Age: neonates < 6 h
- All neonates used in the 7-d survival and reproduction tests were produced by daphnids in culture that had released at least three broods
- Feeding during test
- Food type: mixture of Cerophyl and the green alga, S. capricornutum
- Amount: 120 µg Cerophyl/ml and 6.7x10°s S. capricornutum cells/ml
- Frequency: daily after each 24 h renewal

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Remarks on exposure duration:

In this test the toxicity of nitroguanidine and photolyzed nitroguanidine was determined in a cladoceran 7-d survival and reproduction test.

Post exposure observation period:

not applicable

Hardness:

190 mg/l (as CaCO3)

Test temperature:

25 +/- 1 °C

pH:

7.9 (7.4-8.1)

Dissolved oxygen:

7.9 (7.4-8.1) mg/l

Salinity:

not applicable

Nominal and measured concentrations:

Nitroguanidine:
- Nominal concentrations: 0, 260, 430, 720, 1200, 2000 mg/L
- Mean measured concentrations: 0, 260, 440, 730, 1180, 1400 mg/L
> mean measured concentrations were used in the statistical analyses

Photolyzed nitroguanidine:
- Nominal concentrations: 0, 1.3, 2.2, 3.6, 6.0, 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 50 mL glass beakers filled with 30 mL test solution
- Renewal rate of test solution: once daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of broods per replicate: 3
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: non-chlorinated deep well located at JHU/APL
- Total organic carbon: 19 mg/L
- Metals: all below L.O.D.
- Pesticides: all below L.O.D.
- Chlorine: not chlorinated
- Alkalinity: 156 mg/L (as CaCO3)
- Conductivity: 335 µmhos/cm


OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: fluorescent light; 60-85 foot candles at the surface of the culture vessels


EFFECT PARAMETERS MEASURED: mortality and reproduction

Reference substance (positive control):

no

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

440 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

neonate production

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

260 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

neonate production

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

photolyzed nitroguanidine

Basis for effect:

mortality

Remarks:

of the adults

Remarks on result:

other: effect through photolyzed nitroguanidine

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

photolyzed nitroguanidine

Basis for effect:

mortality

Remarks:

of the adults

Remarks on result:

other: effect through photolyzed nitroguanidine

Details on results:

see tables 1 and 2 below

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- Mean measured concentrations were used in the statistical analyses
- Raw daphnid survival data sets were analyzed by Fisher's Exact test
- Raw daphnid reproduction data sets were analyzed by Chi-Square Test for Normality and Steel's Many-One Rank Test
- The statistical tests were performed using SAS (1979) and Toxstat (Gulley et al., 1989)
- A minimum probability level of 0.05 was used for all tests

Table 1: Cladoceran NQ Chronic Toxicity Data – Survival of Adults, Number of Young produced per Brood, Total Number of Young, and Mean Number of Young per Brood after 7 Days of Exposure

Conc. (mg/L)	Rep	Brood No. 1	Brood No. 2	Brood No. 3	Total Young	Mean Young Per Brood
Control	1	4	0	10	14	7.0
	2	3	6	10	19	6.3
	3	4	8	12	24	8.0
	4	4	8	9	21	7.0
	5	3	9	8	20	6.7
	6	2	6	12	20	6.7
	7	4	7	10	21	7.0
	8	3	8	13	24	8.0
	9	2	6	11	19	6.3
	10	4	6	9	19	6.3
260	1	4	4	10	18	6.0
	2	3	6	12	21	7.0
	3	4	9	9	22	7.3
	4	3	10	8	21	7.0
	5	4	0	13	17	8.5
	6	0	3	14	17	8.5
	7	4	6	11	21	7.0
	8	DEAD
	9	4	2	14	20	6.7
	10	4	4	16	24	8.0
440	1	0	2	6	8	4.0
	2	0	2	7	9	4.5
	3	0	0	4	4	4.0
	4	0	3	4	7	3.5
	5	0	1	6	7	3.5
	6	0	2	6	8	4.0
	7	0	4	5	9	4.5
	8	0	4	0	4	4.0
	9	0	3	6	9	4.5
	10	0	1	4	5	2.5
730	1	0	0	2	2	2.0
	2	0	0	4	4	4.0
	3	0	0	3	3	6.0
	4	0	0	5	5	5.0
	5	0	0	2	2	2.0
	6	0	0	3	3	3.0
	7	0	0	3	3	3.0
	8	0	0	1	1	1.0
	9	0	0	3	3	3.0
	10	0	0	1	1	1.0
1180	1	0	0	0	0	0
	2	0	0	0	0	0
	3	0	0	0	0	0
	4	0	0	0	0	0
	5	0	0	0	0	0
	6	0	0	0	0	0
	7	0	0	0	0	0
	8	0	0	0	0	0
	9	0	0	0	0	0
	10	0	0	0	0	0
1400	1	DEAD
	2	DEAD
	3	DEAD
	4	DEAD
	5	DEAD
	6	DEAD
	7	DEAD
	8	DEAD
	9	DEAD
	10	DEAD

Table 2: Cladoceran photolyzed nitroguanidine*chronic toxicity test – results of Fisher’s Exact Test on adult survival after 7 days of exposure

Concentration of photolyzed nitroguanidine [mg/L]	Number Alive	Number Dead	Critical Value	b Value	Sogn
Control	10	0
1.3	10	0	6	10
2.2	9	1	6	9
3.6	0	10	6	0	*
6.0	0	10	6	0	*
10.0	0	10	6	0	*

* Significantly different at alpha = 0.05

Validity criteria fulfilled:

yes

Remarks:

although this study is conducted in accordance to national standard method the validity criteria according to OECD Guideline 211 are fulfilled.

Conclusions:

Nitroguanidine:
Significant mortality (alpha = 0.05) to the adults occurred at 1400 mg/l (see table 1). Neonate production was reduced (alpha = 0.05) at all concentrations down to 440 mg/l; no effects occurred at 260 mg/l. The LOEC and NOEC for the cladoceran, based on reduction in neonate production , are 440 and 260 mg/l, respectively.
Therefore, it can be concluded that nitroguanidine is practically non-toxic towards the cladoceran under the conditions of this test.

Photolyzed nitroguanidine:
Significant mortality (alpha = 0.05) to the adults occurred at all nominal test concentrations down to 3.6 mg/L (see table 2). No difference in neonate production relative to the controls occurred at 1.3 and 2.2 mg/L photolyzed nitroguanidine. Neonate production from from 3.6 to 10.0 mg/L photolyzed nitroguanidine were excluded from the statistical analysis because 100 % mortality occurred to the adults at these concentrations. The LOEC and NOEC for the cladoceran, based on survival of the adults are 3.6 and 2.2 mg/L, respectively.
Photolyzis increases the toxicity of nitroguanidine towards the cladoceran by a factor > 100.

Executive summary:

The 7-day-chronic toxicity of nitroguanidine to Ceriodaphnia dubia was studied under static renewal conditions. Daphnids were exposed to nitroguanidine at mean measured concentrations of 0, 260, 440, 730, 1180, and 1400 mg/L and to nominal concentrations of photolyzed nitroguanidine at 0, 1.3, 2.2, 3.6, 6.0, and 10.0 mg/L.

For nitroguanidine significant mortality (α= 0.05) to the adults occurred at 1400 mg/L.

Neonate production was reduced (α = 0.05) at all concentrations down to 440 mg/L; no effects occurred at 260 mg/L.

For photolyzed nitroguanidine significant mortality (α= 0.05) to the adults occurred at all nominal test concentrations down to 3.6 mg/L (see table 2). No difference in neonate production relative to the controls occurred at 1.3 and

2.2 mg/L photolyzed nitroguanidine. Neonate production from from 3.6 to 10.0 mg/L photolyzed nitroguanidine were excluded from the statistical analysis because 100 % mortality occurred to the adults at these concentrations.

The nitroguanidine LOEC and NOEC for the cladoceran, based on reduction in neonate production, are 440 and 260 mg/L, respectively. The most sensitive end point was reproduction.

The LOEC and NOEC of photolyzed nitroguanidine for the cladoceran, based on the survival of the adults, were 3.6 mg/L and 2.2 mg/L (nominal concentrations), respectively.

Photolyzed nitroguanidine was approximately two orders of magnitude more toxic to cladoceran than the parent compound under the same test conditions. The environmental fate of nitroguanidine in surface waters has been shown to be dominated by photolysis; thus, photolysis should be considered in an hazard evaluation of nitroguanidine discharged to the aquatic environment.

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Description of key information

The nitroguanidine LOEC (7 d) and NOEC (7 d) for the cladoceran, based on reduction in neonate production (most sensitive endpoint), are 440 and 260 mg/L, respectively. The LOEC (7 d) and NOEC (7 d) of photolyzed nitroguanidine for the cladoceran, based on the survival of the adults, were 3.6 mg/L and 2.2 mg/L (nominal concentrations), respectively. Photolyzed nitroguanidine was approximately two orders of magnitude more toxic to cladoceran than the parent compound under the same test conditions. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

2.2 mg/L

Additional information

The 7-day-chronic toxicity of nitroguanidine to Ceriodaphnia dubia was studied under static renewal conditions. Daphnids were exposed to nitroguanidine at mean measured concentrations of 0, 260, 440, 730, 1180, and 1400 mg/L and to nominal concentrations of photolyzed nitroguanidine at 0, 1.3, 2.2, 3.6, 6.0, and 10.0 mg/L.

For nitroguanidine significant mortality (α= 0.05) to the adults occurred at 1400 mg/L.

Neonate production was reduced (α = 0.05) at all concentrations down to 440 mg/L; no effects occurred at 260 mg/L.

For photolyzed nitroguanidine significant mortality (α= 0.05) to the adults occurred at all nominal test concentrations down to 3.6 mg/L. No difference in neonate production relative to the controls occurred at 1.3 and 2.2 mg/L photolyzed nitroguanidine. Neonate production from from 3.6 to 10.0 mg/L photolyzed nitroguanidine were excluded from the statistical analysis because 100 % mortality occurred to the adults at these concentrations.

The nitroguanidine LOEC and NOEC for the cladoceran, based on reduction in neonate production, are 440 and 260 mg/L, respectively. The most sensitive end point was reproduction.

The LOEC and NOEC of photolyzed nitroguanidine for the cladoceran, based on the survival of the adults, were 3.6 mg/L and 2.2 mg/L (nominal concentrations), respectively.

Photolyzed nitroguanidine was approximately two orders of magnitude more toxic to cladoceran than the parent compound under the same test conditions.

Table 1: Study synopsis

Guideline Test Type	Species	Effect Concentration (mg/L)	Reference	Reliability
Goodman, 1986. Proposed new standard guide for conducting early life-stage toxicity tests with fishes.Draft No. 10. American Society for Testing and Materials.	Ceriodaphnia dubia	Nitroguanidine: LOEC (7d) = 440 mg/L NOEC (7d) = 260 mg/L photolyzed Nitroguanidine: LOEC (7d) = 3.6 mg/L NOEC (7d) = 2.2 mg/L	Burton, D.T.; Peters, G.T.; University of Maryland; 1993	Reliable with restrictions