Cyanoguanidine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - October 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: national Standard Method (Committee on Methods for Toxicity Tests with Aquatic Organisms, 1975)

GLP compliance:

yes

Remarks:

The study was conducted following the intent of the GLP Regtulations and the final report was reviewed by Analytical Bio-Chemistry Laboratories' Qulity Assurance Unit.

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- the definitive test concentrations were obtained by trasnferring appropriate weights of test compound directly to the test chambers
- Before addition to the test chambers, 1.5 ml dimethylformamide (DMF) was added to each sample weight to increase dispersion of the compound in the dilution water
- All test concentrations were based on the total compound, i.e., not corrected for sample purity
- The solvent control chamber received a 1.5 ml aliquot of DMF, which was equivalent to the highest amount used in any test solutions

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: bluegill sunfish
- Source: Osage Catfisheries, Osage Beach, Missouri
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean): 20 (+/- 0.82) mm
- Weight at study initiation (mean and range): 0.19 (+/- 0.031) g
- test chamber loading biomass for definitve test: 0.13 g/l
- Method of breeding:
- Feeding during holding period: standard commercial fish food occassionally supplemented with brine ahrimp nauplii (Artemia sp.) daily until 48-96 h prior to testing
- Feeding during test: none
- All test fish were held in culture tanks on a 16 hour daylight photoperiod and observed at least 14 days prior to testing
- Fish were obsereved daily during the holding period


ACCLIMATION:
- to dilution water and test temperature: 48-96 h prior to testing
- no feeding during acclimation

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period

Hardness:

40-45 mg/l as CaCO3

Test temperature:

22 (+/- 1.0) °C

pH:

initial: 7.2-7.6
during test: 7.1-7.5

Dissolved oxygen:

7.2 - 9.4 mg/l (82 and 107 % saturation)

Salinity:

No data

Nominal and measured concentrations:

only nominal concentrations: 62.5, 125, 250, 500 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: five gallon glass vessels containing 15 L of soft reconstituted water composed of the following compounds in the amounts stated per L of deionized water:
- 40 mg NaHCO3
- 30 mg CaSO4*2 H2O
- 30 mg MgSO4
- 2 mg KCl
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.13 g/l
- based on results of preliminary testing 5 concentrations of the test compound, ranging in a geometric series from 62.5 to 1000 mg/l


RANGE-FINDING TEST:
- duration: 96 h
- preliminary test concentration: 100 mg/l

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 30-35 mg/l as CaCO3


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- moratlity and abnormal effects once every 24 h
- any dead individuals were removed ftom the test chambers after each 24-hour observation

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

- The 96-hour NOEC was estimated to be 1000 mg/l, the highest concentration tested, based on the lack of mortality or observed abnormal (sub-lethal) effects.
- An examination of the fish culture and acclimation records for this test indicated that the fish were in good condition for tetsing (Raw Data)

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis of the concentration vs. effect data (generally mortality) was obtained by employing a computerized LC50 program developed by Stephan et al. This program calculated the LC50 statistic and its 95 % confidence limits using the binominal, the moving average, and the probit tests. However, if no mortality occurred or if a dose response could not be demonstrated over a reasonable range (< 37 to > 63 %) and an LC50 and/or its percent confidence limits could not be calculated. Three different methods of analyzing the data were used since no one method of analysis is appropriate for all possible sets of data that may be obtained. The method of calculation selected for presentation in this report was that which gave the narrowest confidence limits for the LC50 although all three models are valid.

Sublethal observations / clinical signs:

Table 1: The Acute Toxicity of LH 21,671C to Bluegill Sunfish (Lepomis macrochirus)

	LC50in mg(l (ppm)
Compound	24 hours	48 hours	96 hours
LH 21,671C^a	> 1000^b	> 1000	> 1000

 

N = 10 fish per concentration

^aBioassay as conducted at 22°C (+/-1.0), mean fish weight and length, 0.19 (+/-0.031) g and 20 (+/-0.82) mm

^bNo mortality occurred in any concentration tested, therefore an LC₅₀could not be calculated.

 

The 96-hour no-observed effect concentration could be estimated at 1000 mg/l, based on the lack of mortality and abnormal effects at the highest concentration tested.

 

All values were rounded to two significant figures following ABC S.O.P. #8.7.

 

LC₅₀calculated using :

(1)    Binomial Method

(2)    Moving Average Method

(3)   Probit Method

Table 2: Mortality Rates and Water Quality Measurements During the Acute Toxicity Test LH 21,671C to Bluegill Sunfish (Lepomis macrochirus)

Nominal Conc. (mg/l)	Percent Mortality			Water Quality
	Hours			0-hours			48 -hours			96-hours
	24	48	96	Temp. °C	D.O.^amg/l	pH^b	Temp. °C	D.O.^amg/l	pH^b	Temp. °C	D.O.^amg/l	pH^b
Control	0	0	0	22	9.4	7.2	23	7.0	7.1	22	7.3	7.1
Solvent Control	0	0	0	22	9.4	7.3	23	8.0	7.2	22	7.3	7.1
62.5	0	0	0	22	9.4	7.5	23	8.0	7.3	22	7.2	7.1
125	0	0	0
250	0	0	0
500	0	0	0
1000	0	0	0	22	9.4	7.5	23	8.3	7.3	22	7.2	7.1

 

^aDissolved oxygen concentration – Dissolved Oxygen Probe (YSI Model 54)

^bpH – pH Probe (Corning Model 476182) used with a Corning Model 1325 pH and mV meter.

 

NOTE: Dissolved oxygen saturation at the test temperatures of 22 and 23 is 8.8 and 8.7 mg/l, respectively.

Validity criteria fulfilled:

yes

Remarks:

Adequate testing conditions.

Conclusions:

96-hour LC50(95 % C.I.) > 1000 mg/l
96-hour NOEC > 1000 mg/l
=> The test substance is practically non-toxic to Bluegill sunfish under the conditions of the test.

Executive summary:

The acute toxicity of LH 21,671C (Dicyandiamide ) to bluegill sunfish (Lepomis macrochirus) was assessed using the methods outlined by the Committee on Methods for Toxicity Tests with Aquatic Organisms. Water quality parameters of temperature, dissolved oxygen and pH were measures throughout the test an were within acceptable limits. Culture and acclimation period records indicated the fish were in good condition for testing.

 

The study was conducted at the following nominal concentrations of LH 21,671C: 62.5, 125, 250, 500 and 1000 mg/l. Ten fish, with a mean weight of 0.19 (± 0.031) g and a mean standard length of 20 (± 0.82) mm, were exposed to each test concentration and control.

 

The results of the four day static fish toxicity study using LH 21,671C are summarized below. The 24- and 48-hour LC₅₀values were also determined.

 

           96-hour LC₅₀(95 % C.I.) > 1000 mg/l

 

Also, the results indicated a 96-hour, no-observed effect concentration could be estimated at 1000 mg/l, which was based on the lack of mortality and abnormal effects at the highest concentration tested.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: national guideline: Committee on Methods for Toxicity Tests with Aquatic Organisms, 1975

Deviations:

yes

Remarks:

see section "details on results"

GLP compliance:

yes

Remarks:

This study was cond. in acc. with EPA/FIFRA GLP Standards, 40 CFR Part 160, effec. May 2, 1984, with the following exceptions: Client-approved protocol did not require Biospherics analyze test solutions. Reported res. are based on nom. conc. of test mat.

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Salmo gairdneri
- Source: Trout Lodge, McMillin, Washington (eyed eggs)
- Age at study initiation: approx. 4 months
- Length at study initiation (mean, range): 39.3 mm (36.1-43.7 mm)
- Weight at study initiation (mean and range): 1.06 g (0.76-1.49g)
- Method of breeding: eggs were placed in incubator at 15 °C; after hatching, the fry were held in the incubator until yolk sacy were nearly resorbed; the young were then transferred to large holding tanks and maintained on a diet of Trout Chow
- No mortalities were observed in the two weeks preceding testing
- Fish were taken off feed 48 hours before use
- Feeding during test: none


ACCLIMATION
- Acclimation period: two weeks preceding testing
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Trout chow
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no mortality

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

115 ppm as CaCO3

Test temperature:

13 °C

pH:

7.4-8.0

Dissolved oxygen:

6.8-10.4 ppm

Salinity:

No data

Nominal and measured concentrations:

- range-finding test: 1, 10, 100, 1000, 10000 ppm (nominal)
- definitive test: 1296, 2160, 3600, 6000, 10000 ppm (nominal)

Details on test conditions:

TEST SYSTEM - range-finding test:
- Test vessel: polystyrene tanks (28L x 18W x 13D cm) each containing 4 liters (8cm deep) of dilution water
- Aeration: no
- Test concentrations were prepared by adding known amounts of test material directly to each tank
- Test concentrations (nominal): 1, 10, 100, 1000, 10000 ppm
- dilution water control: container with 100% well water
- No. of organisms per vessel: 3
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1


TEST SYSTEM - definitive test:
- Test vessel: 19 liter glass carboys, each containing 15 liter (28 cm deep) of dilution water
- Aeration: no
- 5 test concentrations were prepared by adding known amounts of test material directly to each carboy
- Test concentrations (nominal): 1296, 2160, 3600, 6000, 10000 ppm
- dilution water control: container with 100% well water
- No. of organisms per vessel: 10 (random assignment)
- Biological loading: ~0.71 g/L
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- pH, dissolved oxygen and temperature were recorded every 24 hours over a 96-hours exposure period


TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: 400' deep weel located at the Biosperics Inc. Laboratory with a quality considered acceptable for bioassay purposes
- Preparation of dilution water: aeration before use
- Alkalinity: 184 ppm as CaCO3
- Conductivity: 360 µmhos/cm


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortalities and any observable abnormal behaviour among the test fish were also noted and were recorded every 24 hours over a 96-hours exposure period
- fish were classified as dead when no movement of fins, gills, or body could be evoked upon disturbance of the organism

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

8 500 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 7000-13000

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

7 700 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 6600-9000

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

3 600 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3 600 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3 600 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3 600 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Although mortality did occur at 2160 ppm, the normal behavior observed at 3600 ppm, as well as the absence of mortalities, led to the selection of the next higher concentration as the no- effect level.
- The single mortality at 2160 ppm appaered to be random, and was discounted in the selction of the no-effect level.
- At all concentrations tested, the test material did not immediately go into solution when added to the dilution water in each container. After 24 h, some compound remained undissolved at the highest concentration tested. At 48 h, dissolution was complete.
- Approx. 100 ml of the test solutions was drawn from each test container at 48 h and 96 h. The samples were stored at 4 °C in ployethylene bottles while awaiting shipment to the sponsor.

PROTOCOL DEVIATIONS:
- pH, alkalinity and conductivity of dilution water used for the range-finding test were below the specified levels
- these deviations will not affect the LC50 determination, whch is based on definitive test results
- the alkalinity of dilution water used in definitive testing was higher than the range specified in the protocol; this deviation shoul not impact significantly onm the test results, since stock animals are acclimated to water of the same quality

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No data

Sublethal observations / clinical signs:

		Time (hrs)
Treatment		24	48	72	96
Control	% dead	0	0	0	0
	behavior	10 – 1i*	10 – 1i	10 – 1i	10 – 1i
10000 ppm	% dead	0	10	70	90
	behavior	10 – 1a	2- 2f, 3b	1 – 2f, 3b	1 – 1b
			3 – 1i	1 – 3b
			4 – 3b	1 – 1i
6000 ppm	% dead	10	10	10	10
	behavior	9 – 1a	9 – 1i	9 – 1i	3 – 1d
					6 – 1i
3600 ppm	% dead	0	0	0	0
	behavior	10 – 1i	10 – 1i	10 – 1i	10 – 1i
2160 ppm	% dead	0	0	0	0
	behavior	10 – 1i	9 – 1i	9 – 1i	9 – 1i
1296 ppm	% dead	0	0	0	0
	behavior	10 – 1i	9 – 1i	10 – 1i	10 – 1i
			1 – 3b

* First number indicates number of animals displaying behavior indetified by second number/letter code

 

Behavioral Response Code
1. General	2. Swimming	3. Pigmentation	4. Integument	5. Respiration
a. Quiescent	a. Ceased	a. Light discolored	a. Muccus shedding	a. Rapid
b. Hyperexcitable	b. Erratic	b. Dark discolored	b. Muccus coagulation	b. Slow
c. Irritated	c. Gyrating	c. Varidscolored	c. Hemorrhagic	c. Irregular
d. Surfacing	d. Skittering			d. Ceased
e. Sounding	e. Inverted			e. Gulping air
f. Twitsching	f. On side			f. Labored
g. Tetanous
h. Flaccid
i. Normal

 

Validity criteria fulfilled:

yes

Remarks:

Validity criteria according to guideline fulfilled.

Conclusions:

The LC50 (96h) of Dicyandiamide to 4 week old rainbow trouts (Oncorhynchus mykiss) was determined to be 7700 ppm based on nominal concentrations. The test was carried out under GLP conditions The NOEC (96h) was 3600 ppm. Therefore, ist can be concluded that Dicyandiamide is practically non-toxic towards 4 week old rainbow trouts under the conditions of this test.

Executive summary:

The acute toxicity of Dicyandiamide to fish was determined according to the method described by the “Committee on Methods for Toxicity Tests with Aquatic Organisms, 1975”. The nominal concentrations tested were 1296, 2160, 3600, 6000, and 10000 ppm. Total exposure duration was four days.

The LC50 (96h) of Dicyandiamide to 4 week old rainbow trouts (Oncorhynchus mykiss)was determined to be 7700 ppm based on nominal concentrations. The test was carried out under GLP conditions The NOEC (96h) was 3600 ppm.

Therefore, it can be concluded that Dicyandiamide is practically non-toxic towards 4 week old rainbow trouts under the conditions of this test.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The acute toxicity of Dicyandiamide was examined for young perch and rouch to various concentrations of the test substance in aerated glass aquaria for 3-4 days.

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

other: Perca fluviatilis and Rutilus rutilus

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

3 d

Remarks on exposure duration:

exposure duration: 3-4 days

Duration:

3 d

Dose descriptor:

LOEC

Effect conc.:

>= 8 000 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: results for perch (young)

Duration:

3 d

Dose descriptor:

LOEC

Effect conc.:

>= 9 000 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: results for rouch (young)

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed

Conclusions:

The threshold value (first observable signs of toxicity) for young perch and rouch in this study was 8000 and 9000 mg/l, respectively.
Therefore, it can be concluded that Dicyandiamide is practically non-toxic to young perch and rouch.

Executive summary:

The acute toxicity of Dicyandiamide was examined for young perch and rouch to various concentrations of the test substance in aerated glass aquaria for 3-4 days.

The threshold value (first observable signs of toxicity) for young perch and rouch in this study was 8000 and 9000 mg/l, respectively.

Therefore, it can be concluded that Dicyandiamide is practically non-toxic to young perch and rouch.

Description of key information

The acute toxicity of Dicyandiamide to fish (Oncorhynchus mykiss, Lepomis macrochirus) was determined according to the method described by the “Committee on Methods for Toxicity Tests with Aquatic Organisms, 1975”. 
The nominal concentrations tested on Oncorhynchus mykiss were 1296, 2160, 3600, 6000, and 10000 ppm. Total exposure duration was four days. 
With Lepomis macrochirus the nominal concentrations tested were 62.5, 125, 250, 500 and 1000 mg/l. 
Nehring (1964) examined the acute toxicity of Dicyandiamide for young perch and rouch to various concentrations of the test substance in aerated glass aquaria for 3-4 days. Test concentrations are not specified. 
No acute toxic effects of Dicyandiamide to fish could be observed under the conditions of the tests applied. 

Additional information

The LC50 (96h) of Dicyandiamide to 4 week old rainbow trouts (Oncorhynchus mykiss) was determined to be 7700 ppm based on nominal concentrations. The test was carried out under GLP conditions The NOEC (96h) was 3600 ppm.

The results of the four day static fish toxicity study using Dicyandiamide are summarized below. The 24- and 48-hour LC₅₀values were also determined.

 

           96-hour LC₅₀(95 % C.I.) > 1000 mg/l

 

Also, the results indicated a 96 h NOEC of 1000 mg/l, which was based on the lack of mortality and abnormal effects at the highest concentration tested.

The threshold value (first observable signs of toxicity) for young perch and rouch in this study was 8000 and 9000 mg/l, respectively.