N-1-naphthylaniline

Toxicity to other aquatic organisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The effects of acute and chronic exposures to the test compounds on viability, growth and metamorphosis were evaluated. Also teratogenic effects were followed. Exposure was accomplished by placing batches of 50 to 100 embryos in 1 litre of aged tap water containg the test substance. Larval stages were tested in groups of 50 to 100 in 100 litre aquaria.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: N-phenyl-alpha-naphthylamine is relatively insoluble in water. In order to bring as much test material in solution as possible and to avoid undissolved particles adhering to embryos and larvae solutions were prepared in the following manner. 2 g of test substance were added to 1L of water. The suspension was agitated on a reciprocating shaker for 24 to 96h and then filtered.

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Rana pipiens

Details on test organisms:

TEST ORGANISM
- Common name: Leopard frog
- Source: purchased from Hazen, Alburg, Vemont
- Method of breeding: Adults maintained at 5°C without feeding. Female R. pipiens were induced to ovulate by injection of pituitary extract. Eggs were inseminated by standard techniques (Rugh, 1962). Embryos (from fertilisation until feeding) and larvae (emerged from external membranes and feeding) were staged acc. to Shumway (1940). Embryos were dejellied in a solution of 3% cysteine, 0.1% papain, pH 8.0 and grown in aged or dechlorinated tap water in 19 cm inner diameter bowls at a density of 100 embryos/L. Post hatching stages were grown in 30 gal all-glas aquaria at a density of one animal per litre. Rana larvae were fed boiled lettuce.

Test type:

static

Water media type:

freshwater

Limit test:

no

Remarks on exposure duration:

no total exposure time given; two test conducted; in one the exposure time was at least 48 h, in a second test until embryo developmental stage 20 was reached

Test temperature:

not specified

pH:

not specified

Dissolved oxygen:

not specified

Nominal and measured concentrations:

20, 200 mg/L (nominal) (suspension in 1st test)
> 5 mg/L (nominal) (solution in 2nd test)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: 1L container (emryos), 100L aquaria (larvae)
- No. of organisms per concentration: 100 and 125
- No. of organisms per control: 100 and 125

Reference substance (positive control):

no

N-phenyl-alpha-naphthylamine is toxic to larvae and embryos of Rana pipiens. In a test with embryos (table 1 below) were exposed to nominal concentrations of 20 and 200 mg/L. Embryos showed no effects until they reached stage 18, a stage which is characterised by motor reactions to external stimuli. If kept in contact with the test substance embryos developed further to stage 20 (characterised by beating heart and blood circulation in external gills) at which point development was arrested, Continued exposure resulted in death.

In a second test larvae were grown in lethal concentrations of the test material for 48 h in concentrations >= 5mg/L. Up to 24 h all larvae survived, wheras within the next 24 h all larvae died.

These results show that embryonic stages are less impermeable for the test substance than the larval stages.

Since the solubility of the substance increases with time and temperature a given amount of the test substance will be more toxic in warm than in cold water and in standing than in running water.

Table 1: Effect of exposure of Rana pipiens embryos to suspensions of N-phenyl-alpha-naphthylamine

Treatment	Number of embryos exposed	Number of embryos surviving
		Stage 10 (dorsal lip)	Stage 14 (neurula)	Stage 18 (muscular response)	Stage 20 (heart beat)
Control	100	100	100	97	90
20 mg/L	100	100	100	100	0
200 mg/L	100	100	100	96	0

Validity criteria fulfilled:

not applicable

Reference 2

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The effects of acute and chronic exposures to the test compounds on viability, growth and metamorphosis were evaluated. Also teratogenic effects were followed. Exposure was accomplished by placing batches of 50 to 100 embryos in 1 litre of aged tap water containg the test substance. Larval stages were tested in groups of 50 to 100 in 100 litre aquaria.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: N-phenyl-alpha-naphthylamine is relatively insoluble in water. In order to bring as much test material in solution as possible and to avoid undissolved particles adhering to embryos and larvae solutions were prepared in the following manner. 2 g of test substance were added to 1L of water. The suspension was agitated on a reciprocating shaker for 24 to 96h and then filtered.

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: South African clawed toad
- Source: captured in drainage ditches in Costa Mesa, California or purchased from South African Snake Farm, Fish Hoek, Cape Province, South Africa
- Method of breeding: Adults maintained in glass aquaria at room temperature and fed Purina Trout Chow twice weekly. Fertilised eggs were obtained by injecting pairs of frogs with human chorionic gonadotropin (Sigma) by standard laboratory techniques (Brown, 1970). Embryos (from fertilisation until feeding) and larvae (emerged from external membranes and feeding) were staged acc. to Nieuwkoop & Faber (1956). Embryos were dejellied in a solution of 3% cysteine, 0.1% papain, pH 8.0 and grown in aged or dechlorinated tap water in 19 cm inner diameter bowls at a density of 100 embryos/L. Post hatching stages were grown in 30 gal all-glas aquaria at a density of one animal per litre. Xenopus larvae were fed yeast.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

168 h

Hardness:

not specified

Test temperature:

not specified

pH:

not specified

Dissolved oxygen:

not specified

Nominal and measured concentrations:

0, 5.6, 6.2, 7.12 mg/L (measured) (solution in 1st test)
100 mg/L (nominal) (solution in 2nd test)
20, 200 mg/L (nominal) (suspension in 3rd test)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: 1L container (emryos), 100L aquaria (larvae)
- No. of organisms per concentration: 36 - 85
- No. of organisms per control: 90

Reference substance (positive control):

no

N-phenyl-alpha-naphthylamine is toxic to larvae and embryos of Xenopus laevis. In a test with larvae (table 1 below) exposure of the larvae to concentrations up to 5.6 mg/L produced no observable effects. Concentrations greater than 5.6 mg/L were teratogenic. The exposure of larvae to this sublethal concentration of N-phenyl-alpha-naphthylamine resulted in retardation of growth and abnormal development. Malformed larvae died before completing metamorphosis. The syndrome of anomalies was complex, affecting the head and trunk. Concentrations >= 6.2 mg/L were lethal with no surviving larvae after 24 h. Swimming activity decreased during the first hour of exposure and ceased by the end of the second hour. Continued exposure led to a gradual decrease in heart rate which eventually became irregular. Death occured within 24 h.

In a second test (table 2 below) larvae were grown in lethal concentrations of the test material for certain time intervals, then transferred into uncontaminated water and survival observed. Survivers usually developed normal, however, the survival rate was inversely proportional to duration of exposure.

In a third test embryos were exposed to nominal concentrations of 20 and 200 mg/L of the test substance and the developmental stages of the embryos was followed. Embryos showed no effects until they reached stage 24, a stage which is characterised by motor reactions to external stimuli. If kept in contact with the test substance embryos failed to develop further.

These results show that embryonic stages are less impermeable for the test substance than the larval stages.

Since the solubility of the substance increases with time and temperature a given amount of the test substance will be more toxic in warm than in cold water and in standing than in running water.

Table1: Effect of exposure to solutions (100 mg/L, 48 h-shaking, room temp., filtered) of amines on viability of Xenopus larvae

	Concentration (mg/L)	Number of larvae exposed	Number of larvae surviving
			24 h	168 h
Control	0	90	84	82
N-phenyl-alpha-naphthylamine	5.6	85	85	80
	6.2	20	0	0
	7.12	36	0	0

Table2: Effect of varying lengths of exposure to N-Phenyl-alpha-Naphthylamine on survival of Xenopus-larvae

Length of exposure	Number of larvae exposed	Number of larvae surviving
		1 day	5 days	12 days	20 days
Control	150	150	147	145	138
12 h	50	41	41	41	41
24 h	150	111	109	97	92
30 h	100	100	13	13	13
36 h	100	100	7	7	0
96 h	100	100	22	19	2

Validity criteria fulfilled:

not applicable

Reference 3

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The objective of this study was to ascertain whether the test compound is a teratogen

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: N-phenyl-alpha-naphthylamine is relatively insoluble in water. In order to bring as much test material in solution as possible and to avoid undissolved particles solutions were prepared in the following manner. The test substance was added to water. The suspension was agitated on a reciprocating shaker for various periods of time and then filtered.

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Rana pipiens

Details on test organisms:

TEST ORGANISM
- Common name: Leopard frog
- Source: purchased from Hazen, Alburg, Vemont
- Method of breeding: Adults maintained at 5°C without feeding. Female R. pipiens were induced to ovulate by injection of pituitary extract. Eggs were inseminated by standard techniques (Rugh, 1962). Embryos (from fertilisation until feeding) and larvae (emerged from external membranes and feeding) were staged acc. to Shumway (1940). Embryos were dejellied in a solution of 3% cysteine, 0.1% papain, pH 8.0 and grown in aged or dechlorinated tap water in 19 cm inner diameter bowls at a density of 100 embryos/L. Post hatching stages were grown in 30 gal all-glas aquaria at a density of one animal per litre. Rana larvae were fed boiled lettuce.

Test type:

static

Water media type:

freshwater

Test temperature:

not specified

pH:

not specified

Dissolved oxygen:

not specified

Nominal and measured concentrations:

20, 200 mg/L (nominal) (suspension)

Details on test conditions:

not specified

Reference substance (positive control):

no

Dose descriptor:

EC50

Effect conc.:

>= 4 - <= 5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

morphology

Exposure to N-phenyl-alpha-naphthylamine resulted in teratogenesis and increased mortality. Of 100 embryos exposed to each of the two concentrations 100 showed malformations, i.e. 100%. Although the relative sensitivity of the eggs to N-phenyl-alpha-naphthylamine varied from clutch to clutch, this compound was teratogenic to all clutches of eggs tested.

All known teratogens have a "critical period" of development during which they exert their toxic effects. Exposure to the compound at other developmental stages may be lethal but rarely leads to congenital defects. N-phenyl-alpha-naphthylamine conforms to this pattern. Embryos exposed during neurulation become malformed. Exposure of earlier or later stages may be toxic but is not teratogenic. The syndrome of malformations induced by exposure of frog neurulae to the test substance typically includes shortening of the trunk and intestinal tract and edema.

Reference 4

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The objective of this study was to ascertain whether the test compound is a teratogen

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: N-phenyl-alpha-naphthylamine is relatively insoluble in water. In order to bring as much test material in solution as possible and to avoid undissolved particles solutions were prepared in the following manner. The test substance was added to water. The suspension was agitated on a reciprocating shaker for various periods of time and then filtered.

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: South African clawed toad
- Source: captured in drainage ditches in Costa Mesa, California or purchased from South African Snake Farm, Fish Hoek, Cape Province, South Africa
- Method of breeding: Adults maintained in glass aquaria at room temperature and fed Purina Trout Chow twice weekly. Fertilised eggs were obtained by injecting pairs of frogs with human chorionic gonadotropin (Sigma) by standard laboratory techniques (Brown, 1970). Embryos (from fertilisation until feeding) and larvae (emerged from external membranes and feeding) were staged acc. to Nieuwkoop & Faber (1956). Embryos were dejellied in a solution of 3% cysteine, 0.1% papain, pH 8.0 and grown in aged or dechlorinated tap water in 19 cm inner diameter bowls at a density of 50-100 embryos/L.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Remarks on exposure duration:

not specified

Test temperature:

not specified

pH:

not specified

Dissolved oxygen:

not specified

Nominal and measured concentrations:

6 mg/L (measured)

Reference substance (positive control):

no

Dose descriptor:

EC50

Effect conc.:

>= 4 - <= 5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

morphology

Exposure to N-phenyl-alpha-naphthylamine resulted in teratogenesis and increased mortality. Of 176 embryos exposed 167 showed malformations, i.e. 94.8%. Although the relative sensitivity of the eggs to N-phenyl-alpha-naphthylamine varied from clutch to clutch, this compound was teratogenic to all clutches of eggs tested.

All known teratogens have a "critical period" of development during which they exert their toxic effects. Exposure to the compound at other developmental stages may be lethal but rarely leads to congenital defects. N-phenyl-alpha-naphthylamine conforms to this pattern. Embryos exposed during neurulation become malformed. Exposure of earlier or later stages may be toxic but is not teratogenic. The syndrome of malformations induced by exposure of frog neurulae to the test substance typically includes shortening of the trunk and intestinal tract and edema.

Validity criteria fulfilled:

not applicable

Reference 5

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Principles of method if other than guideline:

The effects of exposures to the test compounds on viability and teratogenity were observed.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: N-phenyl-alpha-naphthylamine is relatively insoluble in water. In order to bring as much test material in solution as possible and to avoid undissolved particles adhering to embryos and larvae solutions were prepared in the following manner. The dry test substance was added to water. The suspension was agitated on a reciprocating shaker and then filtered.

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: South African clawed toad
- Source: captured in drainage ditches in Costa Mesa, California or purchased from South African Snake Farm, Fish Hoek, Cape Province, South Africa
- Method of breeding: Adults maintained in glass aquaria at room temperature and fed Purina Trout Chow twice weekly. Fertilised eggs were obtained by injecting pairs of frogs with human chorionic gonadotropin (Sigma) by standard laboratory techniques (Brown, 1970). Embryos (from fertilisation until feeding) and larvae (emerged from external membranes and feeding) were staged acc. to Nieuwkoop & Faber (1956). Embryos were dejellied in a solution of 3% cysteine, 0.1% papain, pH 8.0 and grown in aged or dechlorinated tap water in 19 cm inner diameter bowls at a density of 100 embryos/L. Post hatching stages were grown in 100L aquaria at a density of one animal per litre.

Test type:

static

Water media type:

freshwater

Limit test:

no

Remarks on exposure duration:

48h to determine LC50; exposure time to determine teratogenic effects was from blastula stage until hatching

Test temperature:

not specified

pH:

not specified

Dissolved oxygen:

not specified

Nominal and measured concentrations:

1, 1.25, 1.5, 1.75, 2.0, 3.0, 4.0 mg/L (measured) (actute test)
3.0, 4.0, 4.5, 5.3, 6.0, 6.8, 7.5 mg/L (measured) (teratogenicity test)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: 1L container (emryos), 100L aquaria (larvae)
- No. of organisms per concentration: 36 - 85
- No. of organisms per control: 90

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

mortality

Remarks on result:

other: 95% CL: 1.96 - 2.76

Dose descriptor:

EC50

Effect conc.:

4.57 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Basis for effect:

morphology

Remarks:

malformations

Remarks on result:

other: 95% CL: 3.93 - 5.30

Table: Effect of N-Phenyl-alpha-Naphthylamine on viability of Xenopus larvae

Exp. No.	Concentration in mg/L
	1.0	1.25	1.5	1.75	2.0	3.0	4.0
	dead/total
1	0/100	2/100	0/25	13/44	77/77	52/52	5/5
2	0/10	0/10	2/100	0/25	20/30	0/5	10/10
3	0/10	0/10	3/10	2/100	15/27	10/10	10/10
4	7/20	0/10	8/10	0/10	0/5	0/5	1/5
5	0/10	0/11	0/10	8/9	0/5	11/11
6	0/10		0/10		6/10	10/10
7					24/53
Totals	7/160	2/141	13/165	23/188	142/297	83/93	26/30
% Dead	4.3	1.4	7.8	12.2	47.8	89.2	86.6

Table: Teratogenic effect of N-Phenyl-alpha-Naphthylamine on Xenopus embryos (embryos were continuously exposed to this compound from blastula until hatching at which time the number of malformed embryos/total number of embryos exposed was recorded)

Exp. No.	Concentration in mg/L
	3.0	4.0	4.5	5.3	6.0	6.8	7.5
	malformations/total
1	15/100	34/100	100/100	148/292	80/85	17/17	73/73
2		18/105	17/100	116/219	87/91		10/10
3		4/79	41/82	0/50	115/115
4					21/21
Totals	15/100	56/284	158/282	264/561	303/312	17/17	83/83
% Dead	15	20	56	47	97	100	100

Validity criteria fulfilled:

not applicable

Additional information

The toxicity and teratogenic effects of N-phenyl-alpha-naphthylamine to amphibians were investigated in several studies with Rana pipiens and Xenopus laevis (Greenhouse 1976 a,b; 1977). Aqueous exposure resulted in teratogenesis and increased mortality with an 48h-LC50 value of 2.3 mg/L and an 48h-EC50 value of 4.57 mg/L for morphological malformations in Xenopus laevis. Another study yielded a NOEC of 5.6 mg/L. Teratogenic effects were observed at 5.6 mg/L. Among the observed effects were retardation of growth and abnormal development. Concentrations above 6.2 mg/L were lethal after 24 hours.

All known teratogens have a "critical period" of development during which they exert their toxic effects. Exposure to the compound at other developmental stages may be lethal but rarely leads to congenital defects. N-phenyl-alpha-naphthylamine conforms to this pattern. Embryos exposed during neurulation become malformed. Exposure of earlier or later stages may be toxic but is not teratogenic. The syndrome of malformations induced by exposure of frog neurulae to the test substance typically includes shortening of the trunk and intestinal tract and edema. Since the effect values did not show to be lower than the effect values obtained for the standard test organisms, the data are not considered for hazard and risk assesment of the test substance.