1-nitroguanidine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Aquatic Toxicology. 1979. Payne and Hall. A method of measuring algal toxicity and its application to the safety assessment of chemicals. 171-180.

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier Aldrich Chemical Co
Water Content 25%

Analytical monitoring:

yes

Details on sampling:

Details not presented; details are described elsewhere (Burrows et al., 1984. Nitroguanidine Wastewater Pollution Control Technology: Phase II Wastewater Characterization and Analytical Methods Development for Organics. Technical Report 8311, AD A141176. US Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD)

Vehicle:

no

Details on test solutions:

- Algal assay medium was prepared as described by Miller et al.
- For Nitroguanidine and photolyzed nitroguanidine algla toxicity tests, two filter-sterilised stock solutions were prepared ; one stock was algal assay medium used for dilution of the other stocks, which were either nitroguanidine dissolved in algal assay medium or photolyzed nitroguanidine which had been photolyzed in algal assay medium
- Test solutions were prepared by dispensing appropriate amounts of the stocks into the DeLong flasks under sterile conditions in a Labconco tissue culture enclosure
- An inoculum od S. capricornutum in long-phase growth was added to give an initial cell count of 20000 cell/ml (+/- 10%)


TEST WITH PHOTOLYZED NITROGUANIDINE:
- 5 test concentrations were used so that an LC50 or EC50 could be adequately defined
- Photolyzed nitroguanidine was prepared by exposing nitroguanidine in an appropriate solution (algal assay meidum) to UV light until the nitroguanidine concentration reached non-detectable levels ( < 0.1 mg/l), as determined by HPLC
- The UV light used was a 450 Watt Canrad-Nanovia Model L quartz mercury-vapor immersion lamp; of the total radiated energy, about 31 watts (17.6%) was contributed by wavelengths shorter than those found in natural sunlight (less than 294 nm)
- nominal concentrations were used in calculations

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): original algal stock solution obtained from the American Type Culture Collection (No. 22662), Docksville, MD
- Method of cultivation: maintained under cool white fluorescent lights (4300 lux +/- 10 %)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 d

Post exposure observation period:

not applicable

Hardness:

no data

Test temperature:

24 +/- 1 °C

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

- Mean measured concentrations, nitroguanidine: 3753, 2002, 990, 503, <1.45 (control) mg/l
- Mean measured concentrations, photolyzed nitroguanidine: 316, 158, 79, 39.5, 19.7, 9.87, 0 (control) mg/l (given as nitroguanidine)

Details on test conditions:

TEST SYSTEM
- Test vessel: DeLong flasks with stainless with caps (125ml)
- Fill volume: 60 ml (filter-steriled algal assay medium)
- Aeration: no
- Flasks in Psycrothera model G27 incubator-shaker set at a rotational speed of 100 rpm (+/- 10 %)
- Initial cells density: 20000 cells/ml (+/- 10 %)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: algal assay medium was prepared as described by Miller et al.
- Detailed composition if non-standard medium was used:


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: not reported
- Light intensity and quality: cool white fluorescent lights (4300 lux +/- 10 %)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Dry weight
- Chlorophyll a measurement

Reference substance (positive control):

no

Key result

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

ca. 2 000 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: standing crop dry weight and chlorophyll a levels

Key result

Duration:

5 d

Dose descriptor:

EC10

Effect conc.:

ca. 160 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

standing crop dry weight

Remarks on result:

other: Values not given in test report but calculated on basis of data reported.

Key result

Duration:

5 d

Dose descriptor:

EC10

Effect conc.:

ca. 32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll a levels

Remarks on result:

other: Values not given in test report but calculated on basis of data reported.

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

ca. 32.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

photolyzed nitroguanidine

Basis for effect:

other: standing crop dry weight

Duration:

5 d

Dose descriptor:

EC10

Effect conc.:

ca. 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

photolyzed nitroguanidine

Basis for effect:

biomass

Remarks on result:

other: Values not given in test report but calculated on basis of data reported.

Details on results:

- At the top concentration, which was the approximate saturation concentration of nitroguanidine in algal assay medium at 24 °C, no algicidal or algiostatic effects were observed
- A 120 h EC50 based on growth rates could not be calculated since neither dry weight nor chlorophyll a growth rates were reduced below 50 % of the control levels
- Exponential growth in the control (for algal test): yes
- A 120 h EC50 based on standing crop dry weight and relative chlorophyll a levels could be calculated and was found to be approx. 2000 mg/l for both dry weight and relative chlorophyll a measurements
- Both growth end points were significantly different from controls at all nitroguanidine concentrations tested, so an NOEC could not be determined


Photolyzed nitroguanidine:
- Analysis of dry weight and chlorophyll a data (see tables 5 and 6) showed that neither algicidal nor algistatic effects were obtained. Growth rate EC50’s could not be computed due to non-linear growth rates at high treatment levels.
- Large and significant reductions in both growth parameters at all concentrations above 19.7 mg/l
- The level of toxicity (see table 8) is much higher than that observed for S. capricornutum and nitroguanidine (see table 4)

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- A log(10) transformation was used for all growth measurements and, when a single point estimate of a treatment effect was required, the geometric mean of three replicate flasks was used. A 0.05 significance was used, and significance levels were adjusted for simultaneity using Bonferroni's correction when appropriate.
- The minimum algistatic concentration was determined by an inverse regression technique described by Payne and Hall, 1979
- EC50 based on growth rate: trends were analyzed using a multivarita analysis of variances approach; effects were tested using SAS MANOVA procedure; Kharti's techniques were then used to to fit the concentration-response data to a polynomial model
- EC50 based on 5-day standing crop: 5-day dry weight and relative chlorophyll a data were modeled using the SAS General Linear Models program
-NOEC: For overall growth rate trends, Kharti's techniques were used in conjunction with a multivariate analysis of variance to provide pairwise tests between treatments and the controls; a classical analysis of variance was used to compute 5-day standing crop NOEC's; in this case, the SAS ANOVA procedure was utilized

Table 1: Growth rates of S. capricornutum exposed to nitroguanidine

Mean measured concentration (mg/l)	Growth rate^a
	Dry weight	Relative chlorophyll a
3753	1.50 (40.9)^b	1.45 (-43.7)
2002	2.26 (11.0)	2.19 (-14.9)
990	2.47 (2.8)	2.40 (-6.8)
508	2.46 (3.1)	2.44 (-5.1)
< 1.45 (control)	2.54	2.58

^aMultiplicative change per day. Geometric means of three replicate flasks are reported

^bPercent difference from control levels

Table 2: Effect of Nitroguanidine on the growth of S. capricornutum: dry weight measurements

Mean measured concentration (mg/l)	Test day^a
	0	1	2	5
3753	0.315^b	0.597	0.853	2.92^c(-92.9)^d
2002	0.347	0.811	2.36	22.8^c(-44.4)
990	0.361	0.875	2.80	35.4^c(-13.7)
508	0.323	0.939	2.82	35.9^c(-12.4)
< 1.45 (control)	0.365	0.917	2.90	41.0

^aNo data available for test days 3 and 4

^bGeometric mean of three replicate flasks; mg/l

^cSignificantly different from the controls (p < 0.05). Only day 5 data was tested.

^dPercent difference from control levels

Table 3: Effect of Nitroguanidine on the growth of S. capricornutum: relative chlorophyll a measurements

Mean measured concentration (mg/l)	Test day^a
	0	1	2	5
3753	21.8^b	40.7	86.2	211^c(-93.6)^d
2002	22.9	53.0	175	1600^c(-51.8)
990	22.3	57.7	210	2500^c(-24.7)
508	22.7	61.0	210	2670^c(-19.6)
< 1.45 (control)	22.3	54.5	212	3320

^aNo data available for test days 3 and 4

^bGeometric mean of three replicate flasks; mg/l as corproporhyrin

^cSignificantly different from the controls (p < 0.05). Only day 5 data was tested.

^dPercent difference from control levels

Table 4: Summary of nitroguanidine toxicity to S. capricornutum

End point	Growth Measurement
	Dry weight^a	Relative chlorophyll a^b
Minimum algicidal concentration	> 3753	> 3753
Minimum algistatic concentration	> 3753	> 3753
120 h E50 (growth rate)	> 3753	> 3753
120 h E50 (standing crop)	2146 (2010-2282)^d	1937 (1734-2140)
120 h NOEC	< 508^e	< 508

^amg/l

^bmg/l as corproporhyrin

^cHighest concentration tested (approximate nitroguanidine saturation concentration at 24 °C)

^d95 % confidence limits

^elowest concentration tested

Table 5: Effect of photolyzed Nitroguanidine on the growth of S. capricornutum: dry weight measurements

Nominal^aconcentration	Test Day
	0	1	2	3	4	5
316	0.319^b	0.365	0.469	0.464	0.575	0.465 (-98.7)^c
158	0.294	0.428	0.611	0.762	0.849	0.762 (-97.9)
79.0	0.277	0.558	1.050	1.620	2.641	3.398 (-90.8)
39.5^d	0.286	0.307	0.335	0.346	0.384	0.438 (-98.9)
19.7	0.286	0.740	2.793	8.776	24.148	36.601 (-0.6)
9.87	0.312	0.704	2.839	8.661	23.631	38.494 (+4.5)
0 (control)	0.302	0.752	2.463	7.213	23.554	36.829

^aConcentrations are reported as nitroguanidine (mg/l) prior to photolyzation.

^bGeometric mean of three replicate flasks; mg/l

^cPercent difference from control levels (day 5)

^dThis treatment was excluded from the data analysis

Table 6: Effect of photolyzed Nitroguanidine on the growth of S. capricornutum: chlorophyll a measurements

Nominal^aconcentration	Test Day
	0	1	2	3	4	5
316	18.42^b	26.98	40.72	47.60	54.31	54.30 (-98.2)^c
158	16.32	24.19	41.45	56.86	73.06	85.18 (-97.1)
79.0	17.94	33.63	49.35	153.04	274.12	388.58 (-86.9)
39.5^d	17.00	18.49	19.84	22.22	30.52	40.45 (-98.6)
19.7	17.49	46.72	212.34	785.70	1721.54	2972.56 (+0.6)
9.87	16.66^e	43.81^e	212.85	784.07^e	1760.63	2828.97 (-4.3)
0 (control)	17.26	46.65	176.66	621.17	1601.98	2936.18

^aConcentrations are reported as nitroguanidine (mg/l) prior to photolyzation.

^bGeometric mean of three replicate flasks; mg/l as corproporhyrin

^cPercent difference from control levels (day 5)

^dThis treatment was excluded from the data analysis.

^eGeometric mean of two observations only.

 

Table 8: Summary of photolyzed nitroguanidine toxicity to S. capricornutum

End point	Growth Measurement
	Dry weight^a	Relative chlorophyll a^b
Minimum algicidal concentration	> 316^c	> 3753
Minimum algistatic concentration	> 316	> 3753
120 h E50^d (standing crop)	32.3 (26.2-38.5)^e	34.3 (26.3-42.3)
120 h NOEC Growth rate Standing crop	19.7 19.7	19.7 19.7

^amg/l

^bmg/l as corproporhyrin

^cHighest concentration tested (mg/l); all concentrations reported as nitroguanidine prior to photolyzation

^dGrowth rate EC₅₀was not available to non-linear growth rates at some treatment levels

^e95 % confidence limits

Table 9: The effect of photolyzation on the acute toxicity of nitroguanidine

Species	End point	Toxicant concentration (mg/l)		Relative toxicity ratio
		Nitroguanidine	Photolyzed Nitroguanidine
Algae (S. capricornutum)	120 h EC50	2146 (2010-2283)	32.3 (26.2-38.5)	66.4

 

 

 

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results, nitroguanidine appears to present little hazard to S. capricornutum except at concentrations approaching its solubility limit in water.

Executive summary:

In a 120 h acute toxicity study, the cultures of S. capricornutum were exposed to nitroguanidine at measured test concentrations of 3753, 2002, 990, 503, <1.45 (control) mg/l. In addition, these algae were exposed to photolyzed nitroguanidine at measured concentrations of 316, 158, 79, 39.5, 19.7, 9.87, 0 (control) mg/l (given as nitroguanidine). Tests were conducted under static conditions.

The 120 h EC₅₀(nitroguanidine) values based on standing crop dry weight and chlorophyll a measurements is 2146 mg/l and 1937 mg/l, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 12.4 to 92.9 and 4.3 to 98.2 based on dry weight and chlorophyll a measurements, respectively. 

The 120 h EC₅₀for photolyzed nitroguanidine, based on standing crop dry weight and chlorophyll a measurements, is approximately 32.3 mg/l. The % growth inhibition in the treated algal culture as compared to the control ranged from 0.6 to 98.7 and 19.6 to 93.6 based on dry weight and chlorophyll a measurements, respectively. 

Based on the results of this study, Nitroguanidine appears to present little hazard to S. capricornutum except at concentrations approaching its solubility limit in water.

Photolysis increased the acute toxicity by a factor of 66.4.

Description of key information

The 120 h EC₅₀ (nitroguanidine) values, based on standing crop dry weight and chlorophyll a measurement, is 2146 mg/L and 1937 mg/L, respectively. The 120 h EC50 for photolyzed nitroguanidine, based on standing crop dry weight and chlorophyll a measurement, is approximately 32.3 mg/L. Based on the results of this study, nitroguanidine appears to present little hazard to Raphidocelis subcapitata, formerly known as Selenastrum capricornutum except at concentrations approaching its solubility limit in water. Photolysis increased the acute toxicity by a factor of 66.4.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.3 mg/L

EC10 or NOEC for freshwater algae:

20 mg/L

Additional information

In a 120 h acute toxicity study, the cultures of Selenastrum capricornutum were exposed to nitroguanidine at test concentrations of 3753, 2002, 990, 503, <1.45 (control) mg/l. In addition, algae were exposed to photolyzed nitroguanidine at concentrations of 316, 158, 79, 39.5, 19.7, 9.87, 0 (control) mg/l (given as nitroguanidine). Tests were conducted under static conditions.

The 120 h EC50 (nitroguanidine) values based on standing crop dry weight and chlorophyll a measurements is 2146 mg/L and 1937 mg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 12.4 to 92.9 and 4.3 to 98.2 based on dry weight and chlorophyll a measurements, respectively. The 120h EC10 (nitroguanidine), calculated from the data reported, is ca. 160 mg/L.

The 120 h EC50 for photolyzed nitroguanidine, based on standing crop dry weight and chlorophyll a measurements, is approximately 32.3 mg/L. The % growth inhibition in the treated algal culture as compared to the control ranged from 0.6 to 98.7 and 19.6 to 93.6 based on dry weight and chlorophyll a measurements, respectively. The 120h EC10 (photolyzed nitroguanidine), calculated from the data reported, is ca. 20 mg/L (based on biomass).

Based on the results of this study, nitroguanidine appears to present little hazard to S. capricornutum except at concentrations approaching its solubility limit in water.

Photolysis increased the acute toxicity by a factor of 66.4.

Table 1: Study synopsis

Guideline Test Type	Species	Effect Concentration	Reference	Reliability
Aquatic Toxicology. 1979. Payne and Hall. A method of measuring algal toxicity and its application to the safety assessmnet of chemicals. 171-180.	S. capricornutum	Nitroguanidine: EC50(120h) mg/L = 2146   Photolyzed nitroguanidine: EC50(120 h) mg/L = 32.3	Schalie, W.H. van der; U.S.Army Medical Bioengineering Research and Development Laboratory; 1985	Reliable with restrictions