1-nitroguanidine

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

purity of test substance not reported and no detailed information about soil mixture used

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)

GLP compliance:

not specified

Remarks:

not reported; publication

Specific details on test material used for the study:

Test substance supplier not specified

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on preparation and application of test substrate:

- Hydroponic solutions were directly adjusted to the respective nitroguanidine concentrations
- Plants grown in soil culture: 100 mL of either 0, 2, 4, 6, and 8 mM nitroguanidine were directly applied to the soil on alternate days for a total of 5 applications

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: soybean
- Plant family: Fabaceae
- Varieties: Glycine max L. Merr., cvs. Williams (used in hyroponic culture and soil), Washington and Wayne (used in soil only)
- Source of seed:
- Prior seed treatment/sterilization:
- Historical germination of seed (germination of seed lot tested):
- Seed storage:

Species:

other: Festuca arundinacea

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: tall fescue
- Plant family: Poaceae
- Varieties: Festuca arundinacea Schreb., cvs. KY 31, Mozark, Martin
- These species are commonly grown agriculturally near at least one NQ production site.

Species:

other: Bromus inermis

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: smooth bromegrass
- Plant family: Poaceae
- Varieties: Bromus inermis Leyss., cvs. Achenbach, Blair, and Beacon
- Source of seed:
- Prior seed treatment/sterilization:
- Historical germination of seed (germination of seed lot tested):
- Seed storage:
- These species are commonly grown agriculturally near at least one NQ production site.

Test type:

early seedling growth toxicity test

Study type:

laboratory study

Substrate type:

other: hydroponical culture: soybeans; soil mixture: soybeans and grass

Limit test:

no

Total exposure duration:

14 d

Remarks:

14 days: applicable to soybeans and grasses in soil culture; soybeans in hydroponic culture were examined 25 days after germination/11 days after initial treatment

Post exposure observation period:

Not applicable

Test temperature:

- Hydroponic culture, soybeans: 25/20 °C (day/night temperature);
- Soil cultures: 25/20 °C (day/night temperature)

pH:

no data

Moisture:

no data

Details on test conditions:

TEST SYSTEM
- Amount of soil (soil cultures): 600 mL
- Hydroponic culture: 850 mL jars
- No. of plants (retained after thinning > ten days after germination): grasses: two per pot; soybeans: one per pot
- No. of replicates per treatment group: 5
- No. of replicates per control: 5
- Soil cultures were watered weekly with 1/4 strength Hoagland's solution (Hoagland and Arnon 1950)


PROPERTIES OF SUBSTRATE (if soil)
- Maximum water holding capacity (soil mixture): 100 mL/pot


GROWTH CONDITIONS
- Photoperiod: > hydroponic culture: 14 hrs;
> soil cultures: greenhouse conditions
- Light intensity and quality: > hydroponic culture: visible light intensity of 400 µmol (quanta)/(m²s)
- Day/night temperatures: 25/20 °C
- Watering regime and schedules: soil cultures were watered weekly with 1/4 strength Hoagland's solution (Hoagland and Arnon 1950)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hydroponic cultures: plant dry weight, leaf chlorophyll concentration, cation (Ca, Mg, K) concentration
- Soil cultures: * Leaf chlorophyll concentration (tall fescue and smooth bromegrass only; two weeks after initial treatments)
* Plants were harvested after 14 days and tissues seperated as follows:
1) Soybeans: leaf blades, stems including petioles, and washed roots;
2) Grasses: leaves plus culms, and washed roots



TEST CONCENTRATIONS
- Hydroponic culture: 0, 2, or 4 mM nitroguanidine (hyroponic solutions were adjusted to these concentrations 14 days after germination)
- Soil cultures: 0, 2, 4, 6, or 8 mM nitroguanidine solutions (all prepared with 1/4 strength Hoagland's solution); application of 100 mL of test solutions; age of plants: soybeans: 23 days; age of tall fescue and smooth bromegrass plants: 30 days


COMPARISON OF UPTAKE AND METABOLISM OF NITROGUANIDINE
- Between soybean and tall fescue; between hydroponic and soil culture
- After 21 days (soybean, V4 stage) and 30 days (tall fescue) the soil or hydroponic solutions were adjusted to either 6.5 x 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3, or 5 x 10^-3 M 14C-nitroguanidine solution
- Each pot or jar received 3.70 x 10^4 Bq 14-C nitroguanidine
- Soil grown plants received a single 100 ml application of the nitroguanidine treatment as a root drench
- Hydroponic and soil media without plants were included as controls

Nominal and measured concentrations:

- Nitroguanidine nominal concentration: 0, 2, 4, 6, 8 mM
- Radiolabelled (14C) nitroguanidine solution: 6.5 x 10^-8, x 10^-7, x 10^-6, x 10^-5, x 10^-4, x 10^-3, or 5 x 10^-3 M

Reference substance (positive control):

no

Species:

Glycine max (G. soja)

Duration:

11 d

Dose descriptor:

LOEC

Effect conc.:

2 other: mM

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reduction of soybean dry matter, leaf chlorphyll concentration , and Mg, as well as increase of K

Remarks on result:

other: plants grown in hydroponic solution

Key result

Species:

Glycine max (G. soja)

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

2 other: mM

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reduction of dry matter

Remarks on result:

other: soybean grown in soil

Key result

Species:

other: Festuca arundinacea

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

>= 4 other: mM

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reduction of Ca concentration of leaves

Remarks on result:

other: tall fescue grown in soil

Details on results:

SOYBEANS IN HYDROPONIC CULTURE:
- Leaf chlorophyll concentration was determined 25 days after germination (treatment with NQ started 14 days after germination)
- Plants were also harvested after 25 days after germination, oven-dried at 60 °C for two days, and weighed
- Ground leaf samples (30 mesh) were digested with nitric acid and perchloric acid and cation (Ca, Mg; and K) concentration determined using atomic absorption spectrometry; four replications (one jar each) were used
- Soybean dry matter and conc. of leaf chlorophyll and Mg were reduced by 2 and 4 mM NQ in hydroponic culture; the NQ-induced chlorosis was distributed throughout the leaf but was more prominent at the leaf margins
- Ca conc. was reduced by 4 mM NQ but not by 2 nM NQ and K conc. was increased by 2 mM NQ


SOYBEANS AND GRASSES IN SOIL CULTURE:
- Leaf chlorophyll concentration (tall fescue and smooth bromegrass only) was determined two weeks after initial treatments
- Plants were harvested after 14 days and tissues seperated as follows:
1) Soybeans: leaf blades, stems including petioles, and washed roots;
2) Grasses: leaves plus culms, and washed roots
- Plants were oven-dried at 60 °C for two days, and weighed
- Ground leaf samples from Kentucky 31 tall fescue and Achenbach smooth bromegrass were analyzed for Ca, Mg; and K) concentration as described previously
- In all soil genotypes within a given species responded similarly to NQ tretament and all data within a species were averaged across genotypes
- Soybeans produced less dry matter as the concentration increased from 0 to 8 mM NQ
- The average dry matter of untreated tall fescue plants was 0.62 g/plant, and was significantly reduced only by the 8 mM NQ (0.51 g/plant)
- Dry matter accumulation by smooth bromegrass was unaffected by NQ treatment
- Ca conc. of both tall fescue and smooth bromegrass levels was reduced by 100 ml applications of 4 and 8 mM NQ to the soil
- There was no significant effect of NQ on leaf chlorophyll, Mg, or K conc. of the grasses


COMPARISON OF UPTAKE AND METABOLISM OF NQ
- After 96 h, aliquots (2,0 ml) were obtained from hydroponic solution and and the quantity of 14C determined using scintillation spectrometry
- The quantity of 14C from aliquots of soil (200 mg air dried) was determined by both scintillation spectrometry after methanol extraction and sample combustion
- Plants were harvested, broken into parts, freeze-dried, and weighed
- Ground samples (200 mg aliquots) were oxidized and the quantity of 14C determined
- Metabolism and degradation compounds of NQ were examined after extraction with methanol:water (80:20, v/v) by thin layer chromatography using butanol, ethanol, and water (66:17:17, v/v/v) on cellulose plates
- The quantity of 14C recovered from oxidation of plant material was converted to NQ-equivalents since radioactivity of leaf extracts (Rf=0.41) co-chromatographed with standard NQ (rf=0.43)
- Concentration of NQ-equivalents in rootes and leaves of both species was significantly greater from plants grown in hydroponic media than those grown in soil
- Incomplete and variable recovery of 14C NQ from air-dried soil 96 hrs after 14C NQ solutions were added to soil in the absence of plants
- Complete recovery from plant free hydroponic media
- Apparent loss of NQ from the soil could have reduced the NQ concentration in the soil solution and contributed to the lower tissue NQ concentrations of soil grown plants compared to that of hydroponically grown plants
- The uptake of NQ from both media was passive for both tall fescue and soybean as demonstrated by the linear relationship between total NQ recovered per g root and external NQ concentration
- Extracts of radiolabelled leaves and roots did not indicate the presence of any metabolite in either species
- Despite the apparent absence of NQ metabolism, total plant and hydroponic media 14C-NQ did not account for all 14C initially appplied -> suggests that a plant mediated loss of 14C NQ (or derived but undetected metabolite) occurred from both species -> this loss of C from NQ suggests transformation of NQ into volatile compounds by the plant (loss in soybeans < loss in tall fescue)

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Tests for significance / significant differences between plant species, test concentrations and culture media; no details reported

Validity criteria fulfilled:

not applicable

Conclusions:

Soybean depleted more nitroguanidine from hydroponic solution, accumulated more nitroguanidine per g root biomass, and had greater leaf nitroguanidine concentration than did tall fescue. Despite these observations, there is evidence that the greater toxicity of nitroguanidine to soybean as compared to tall fescue is primarily due to soybean's greater sensitivity to nitroguanidine and not due to its greater uptake and accumulation of nitroguanidine. Soybean leaves were chlorotic with foliar nitroguanidine concentrations of 40.3 µmol/h whereas tall fescue leaves remained green with nitroguanidine concentrations of 78.4 µmol/g. Therefore, it is apparent that the differential toxicity between species is due to differential sensitivity and not due to soybean's greater accumulation and greater leaf nitroguanidine concentrations exhibited at each nitroguanidine concentration.

Executive summary:

Objective of the study was to determine the effects of nitroguanidine to test the toxicity of and to characterize the uptake of nitroguanidine in plants.

Nitroguanidine was applied to soybeans (Glycine max) in hydroponic culture at concentrations of 0, 2, or 4 mM nitroguanidine as well to soybeans and grasses (Festuca arundinacea) grown in soil treated with 100 mL of either 0, 2, 4, 6, and 8 mM nitroguanidine solutions by applying the solution directly to the soil on alternate days for a total of 5 applications.

 

Results for soybeans in hydroponic culture:

Dry weight, leaf chlorophyll concentration, and cation (K, Mg, Ca) concentration was determined after 14 days of treatment. Soybean dry matter and conc. of leaf chlorophyll and Mg were reduced by 2 and 4 mM nitroguanidine in hydroponic culture; the nitroguanidine-induced chlorosis was distributed throughout the leaf but was more prominent at the leaf margins. Ca concentration was reduced by 4 mM nitroguanidine but not by 2 nM nitroguanidine and K conc. was increased by 2 mM nitroguanidine.

 

Results for soybeans and grasses in soil culture:

Dry weight, leaf chlorophyll concentration, and cation (K, Mg, Ca) concentration (tall fescue and smooth bromegrass only) was determined two weeks after initial treatment.

In soil all genotypes within a given species responded similarly to nitroguanidine treatment and all data within a species were averaged across genotypes.

Soybeans produced less dry matter as the concentration increased from 0 to 8 mM nitroguanidine.

The average dry matter of untreated tall fescue plants was 0.62 g/plant and was significantly reduced only by the 8 mM nitroguanidine (0.51 g/plant). Dry matter accumulation by smooth bromegrass was unaffected by nitroguanidine treatment. Ca concentration of both tall fescue and smooth bromegrass levels was reduced by 100 mL applications of 4 and 8 mM nitroguanidine to the soil. There was no significant effect of nitroguanidine on leaf chlorophyll, Mg, or K concentration of the grasses.

 

 

COMPARISON OF UPTAKE AND METABOLISM OF NITROGUANIDINE

Concentration of nitroguanidine-equivalents in roots and leaves of both species was significantly greater from plants grown in hydroponic media than those grown in soil. Incomplete and variable recovery of 14C nitroguanidine from air-dried soil 96 hrs was obtained after 14C nitroguanidine solutions were added to soil in the absence of plants. A complete recovery from plant free hydroponic media could be obtained. The apparent loss of nitroguanidine from the soil could have reduced the nitroguanidine concentration in the soil solution and contributed to the lower tissue nitroguanidine concentrations of soil grown plants compared to that of hydroponically grown plants.

The uptake of nitroguanidine from both media was passive for both tall fescue and soybean as demonstrated by the linear relationship between total nitroguanidine recovered per g root and external nitroguanidine concentration.

Extracts of radiolabeled leaves and roots did not indicate the presence of any metabolite in either species.

Despite the apparent absence of nitroguanidine metabolism, total plant and hydroponic media 14C-nitroguanidine did not account for all 14C initially applied. This suggests that a plant mediated loss of 14C nitroguanidine (or derived but undetected metabolite) occurred from both species. Therefore, this loss of C from nitroguanidine suggests transformation of nitroguanidine into volatile compounds by the plant (loss in soybeans < loss in tall fescue).

 

Soybean depleted more nitroguanidine from hydroponic solution, accumulated more nitroguanidine per g root biomass, and had greater leaf nitroguanidine concentration than did tall fescue. Despite these observations, there is evidence that the greater toxicity of nitroguanidine to soybean as compared to tall fescue is primarily due to soybean's greater sensitivity to nitroguanidine and not due to its greater uptake and accumulation of nitroguanidine. Soybean leaves were chlorotic with foliar nitroguanidine concentrations of 40.3 µmol/g whereas tall fescue leaves remained green with nitroguanidine concentrations of 78.4 µmol/g. Therefore, it is apparent that the differential toxicity between species is due to differential sensitivity and not due to soybean's greater accumulation and greater leaf nitroguanidine concentrations exhibited at each nitroguanidine concentration.

Description of key information

Nitroguanidine was applied to soybeans (Glycine max) in hydroponic culture at concentrations of 0, 2, or 4 mM nitroguanidine as well to soybean and the grass tall fescue (Festuca arundinacea) grown in soil treated with 100 mL of either 0, 2, 4, 6, and 8 mM nitroguanidine solutions by applying the solution directly to the soil on alternate days for a total of 5 applications.

Additional information

After 14 d the application of 2 mM (and higher concentrations) nitroguanidine to soybeans in hydroponic culture resulted in a reduction of dry matter, leaf chlorophyll concentration and Mg concentration. Soybeans grown in soil culture displayed effects only on dry weight at the same concentration.

The application of nitroguanidine lead to a reduction of dry matter in Tall fescue grown in soil culture at a concentration of 8 mM, whereas the application of nitroguanidine had no effect on Smooth bromegrass (Bromus inermis) dry matter at 8 mM. Both grass species displayed effects on the leaf Ca concentration at nitroguanidine concentrations of 4 and 8 mM.

Soybean leaves were chlorotic with foliar nitroguanidine concentrations of 40.3 µmol/g whereas Tall fescue leaves remained green with nitroguanidine concentrations of 78.4 µmol/g. Therefore, it is apparent that the differential toxicity between species is due to differential sensitivity.