Amides, tall-oil fatty, N-[3-(dimethylamino)propyl]

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:

96 hour

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

carried out to the bulk approach see below

Principles of method if other than guideline:

Due to the strong adsorption of the test chemical to organic matter chemical analysis of the test chemical was conducted at the start of the test
and directly after the solution replacement only due to analysis after 48 hours not being technically possible due to the properties of the test
substance. The bulk approach principle requires accurate initial quantification of the test chemical added to the test system and determination of possible loss to glass ware. Based on the principle that the test chemical is stable (ie not hydrolizable or biodegradable) in the time frame between
solution refreshments subsequent adsorption to organic material is accepted as this occurs to a greater extent environmentally. Solution
replenishment takes place to ensure a realistic worst case has been tested and the test is conducted in natural water compliant with the guideline
recommendations for a suitable dilution water.


.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Non Reported

Analytical monitoring:

yes

Details on sampling:

Sampling:

Samples were taken for chemical analysis from the stock and from all concentrations in all new solutions (including those after refreshment) and
once in the old solution of the 1.0 mg/L replicate. The 1.0 mg/L aquarium was also rinsed with leaching solution and this was subsequently
analysed. Samples of 10 mL were initially taken and diluted 1:1 in leaching solution shaken by hand and further diluted (also in leaching solution) if
required to fall within the calibration curve. Chemical analysis was conducted according to the procedure detailed below under analytical methods.

Sample preparation:

For the standards a stock solution of the test substance was made in leaching solution at a concentration of 292 mg/L. This Leaching solution
contained 100 g/L MgCl2 * 6H2O in a Methanol : 2-propanole 50 : 50 (v/v) solution. From this stock solution dilutions were made in the
concentration range 0 – 300 µg/L, for the calibration standards (n = 5). Control standards from the middle range of the calibration series were
analyzed at a minimum rate of one per ten samples and after each test series.

Aqueous samples:
Aqueous samples were diluted 50:50 (v/v) with leaching solution before analysis. Further dilutions if necessary were done in leaching solution.
Samples were filtered over a Pall 0.45 µm GHP membrane filter to remove the suspended solids of the natural water.

Aquarium extraction samples :
In order to determine the amount of test substance adsorbed to the aquarium wall. The test substance was extracted from the aquarium, by rinsed
the walls of the aquarium 3 to 5 times with 100 ml of leaching solution. The resulting solution was subsequently analyzed.

Vehicle:

no

Details on test solutions:

0.0535 g of the test substance was weighed out using the analytical balance for preparation of the stock solution. 80mL of the test medium was added and the solution was sonicated for 2 minutes. The solution was allowed to cool whilst stirring and was made up to 100mL volume using a volumetric flask. A slightly opaque but homogeneous stock without precipitate was observed. The pH of the stock solution was found to be 8.3 and was therefore not adjusted.

Identical procedure was applied with the second stock for solution refreshment after 48 hours except 0.0539 g of the test substance was weighed outand the pH was slightly higher and was therefore adjusted from 8.8 to 8.3.

An appropriate amount of the stock solution required to reach the desired concentration in a total volume of 3 litres was pipetted into each 3 liter
aquarium. The aquarium was then brought up to exactly 3 L volume with a volumetric flask and homogenized with a Teflon coated magnetic stirrer.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test was performed with juvenile Danio rerio (zebra fish), were accepted for use in testing according to laboratory standard operating proceduresand as recommended in the corresponding guideline. The original parent batch was obtained from the “Dieren vriend” in Arnhem the Netherlands .
The fish in stock were fed one to three times per day with commercially available dry, deep frozen food, or fresh Artemia salina nauplii /juvenile
Daphnia magna neonates. Feeding was stopped 24 hours before the test was started, and the animals were not fed during the test. A representative
number of test fish were weighed prior to the test and measured after the test to assess compliance with guideline criteria.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period. Length measurments and physical examination did take place after exposure however.

Hardness:

Total hardness ºdH = <1.

Test temperature:

The temperature in the test room during the test period ranged from 23.1 to 23.75°C.

pH:

The maximum pH variation during the test was from 7.5 to 8.2.

Dissolved oxygen:

The oxygen concentrations varied during the test from 6.9 to 8.4 mg/L.

Salinity:

Not measured conductivity was however 314 µs/cm .

Nominal and measured concentrations:

The nominal test concentrations were 0.2, 0.45, 1.0, 2.2 and 4.4 mg/L including a control.
The measured test concentrations were 0.19,0.42,0.93,2.02 and 4.13 mg/L including a
Due to initial recoveries exceeding 80%. The dosing was considered accurate and nominal concentrations were used for endpoint calculations.

Details on test conditions:

The test was performed as a semi static test which means that the test media was replaced after 48 hours to ensure that a worst case scenario was
tested. The minimum requirement of 7 fish per test concentration (without duplicates) and control were used for ethical reasons.

Under otherwise identical test conditions, the fish were exposed to the chosen concentrations of the test substance as described below and mortality and sub-lethal effects were recorded at least at 30 minutes, 1, 2 and 4 hours after exposure and then and then at approximately 24, 48, 72 and 96 hours.

The fish were considered dead when a lack of opercular movement was observed and touching of the caudal peduncle produced no reaction. Dead fish were removed from the test vessels directly after being observed. In addition to death, sub-lethal effects such as erratic swimming, loss of
reflex, increased excitability, lethargy, changes in physiology, discoloration, pigmentation, excessive mucous production, hyperventilation,
opaque eyes, curved spine or hemorrhaging were recorded if observed.

The fish were randomly placed in the test vessels which were positioned in the test room in a random manner. During the test the vessels were covered with glass plates. The test solutions present in the test vessels were aerated with water-saturated air purified by an active coal filter and a cotton filter during testing.

Reference substance (positive control):

no

Remarks:

Not authorised for ethical reasons

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL = 0.70-1.26 mg/L

Details on results:

Due to a high initial recovery of the initial measured concentrations endpoints were based on Nominal concentrations. Behaviour and mortality of the test organisms is displayed below under other information Table 5. The test substance can be considered as toxic to fish.

Results with reference substance (positive control):

Not permitted by animal testing authority for ethical reasons

Reported statistics and error estimates:

Trimmed Spearman Karber Graphical Method calculated with Toxcalc™ version 5.0.23 .

Sublethal observations / clinical signs:

Analytical method Settings

Table 1

Column:	Agilent Technologies, Zorbax SB-C18 30 x 2.1 mm 3.5 µm
Column temp.:	70 °C
Flow:	0.5 ml/min
Mobile phase:	A= H2O + TFA, NH3and acetic acid B= Methanol: 2-propanol 50:50 (v/v) + TFA, NH3and acetic acid
Gradient mobile phase:	0 min.          95 % A 5 % B   3 min. 0 % A 100 % B   7 min. 0 % A 100 % B   7.1 min. 95 % A 5 % B   9 min. 95 % A 5 % B
Injection volume:	10 µl

Table 2

Ion source:	ElectroSpray
Polarity:	Positive mode
Detector mode:	Multiple Reaction Mode
Delta EMV	300 V
Source parameters:	Gas temperature: 350 °C Gas flow: 12 l/min Nebulizer: 60 psi Capilary voltage: 2000 V

Table 3

Compound	Precursor Ion	MS 1 RES	Product Ion	MS 2 RES	Dwell time (Ms)	Frag. (V)	Collision Energy (V)
C18’ (main component)	367.4	Unit	322.3	Unit	50	160	22
C18’’ *	365.4	Unit	320.3	Unit	50	146	22
C18’’’ *	363.4	Unit	318.3	Unit	50	142	22

* analyzed during the test, however not reported

Analytical method quality criteria

Table 4

Parameter	Limit	Actual
Linearity (R^2)	=0.98	0.999
Repeatability (CV (%)) at lowest standard	= 20	0.6
Repeatability (CV (%)) at highest standard	= 20	1.3
LOQ (µg/L)	<2	0.4
LOD (µg/L)	<2	0.12
System stability (% of nominal)	=10	1.6
Recovery from medium (%)	=70and=110	>90

Table 5

 

Conc. (mg/L)	No. of fish exposed	0 hours		30 mins		1 hour
		No. dead	Effects	No. dead	Effects	No. dead	Effects
0 (Control)	7	-	AN	-	AN	-	AN
0.2	7	-	AN	-	AN	-	AN
0.45	7	-	AN	-	AN	-	AN
1.0	7	-	AN	-	AN	-	RA
2.2	7	-	AN	-	RA	-	RA
4.4	7	-	AN	-	A	2	5R*

 

 

Conc. (mg/L)	No. of fish exposed	2hours		4 hours		24 hours
		No. dead	Effects	No. dead	Effects	No. dead	Effects
0 (Control)		7		-		AN
0.2		7		-		AN
0.45		7		-		AN
1.0	7	-	RA		RA 3*	3	RA
2.2	7	-	A	7*		7
4.4	7	7		7		7

 

 

Conc. (mg/L)	No. of fish exposed	48 hours		72 hours		96 hours
		No. dead	Effects	No. dead	Effects	No. dead	Effects
0 (Control)	7	-	AN		AN		7AN
0.2	7	-	AN		AN		7AN
0.45	7	-	AN		AN		7AN
1.0	7	4	RA	4	RA	4	3 AN
2.2	7	7		7		7
4.4	7	7		7		7

AN = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; RA =Reduced Activity.*=Euthanized for humane reasons

 

Validity criteria fulfilled:

yes

Remarks:

Reliable without restrictions

Conclusions:

The study can be considered reliable and in keeping with the bulk approach without significant limitations. The test substance should be concluded
as very toxic to fish.

Executive summary:

The acute toxicity ofamides rape-oil, N-[3-(dimethylamino) propyl to the fresh water fish Danio rerio(zebra fish) was tested under semi static conditions,with refreshment after 48 hours in natural water for 96 hours in accordance with the OECD and EEC guidelines for testing of chemicals, with minor modifications. The test was performed in compliance with Good Laboratory Practice (GLP).

 

The definitive test was performed with the following nominal test concentrations including a control:

0.2, 0.45, 1.0, 2.2 and 4.4 mg/L

 

The NOEC for the test substance was determined as 0.45 mg/L.

 

The LC₅₀calculated using the Spearman Karber Graphical Method was a 0.94 mg/L

 

Chemical analysis of the initial concentrations of the test chemical was carried out at all concentrations and in the stock solutions. Recovery of the test chemical after 48 hours was not possible due to inherent test chemical properties. Endpoints were therefore based on nominal values confirmed by the initial analytical recoveries of >90% of the nominal concentrations, in line with the bulk approach.

These results are based on the test chemical as described above. No corrections for active ingredient have been made. Endpoints are based on results after 96 hours only.  The study was carried out within the scope of the bulk approach. Chemical analysis of old solutions was not possible due to the strong adsorbing properties of the test substance.

 

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

96 hour

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

no analysis

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

no analysis

Details on test solutions:

Preparation of the stock solution:
The test substance was soluble in water. A stock solution of approximately 10 mg/L of test substance
was prepared as follows: to an accurately measured amount of 0.0100 g of test substance
approximately 150mL of DSW was added. The stock solution was sonicated for about 5 minutes using
a sonics vibracell. Then DSW was added to a final volume of 1 I. A homogenous clear stock solution
was obtained. The pH of the stock solution was 8.1.

Preparation of the test solutions:
The test solutions were prepared by addition of the required amounts of stock solution to the test.
medium to obtain the following concentrations: 0.06 - 0.125 - 0.25 - 0.5 and 1 mg/L. A control
containing only test medium was included in the test.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test was performed with Danio rerio (zebra fish), which were obtained from a retailer and were
accepted according to the quality control criteria of the guidelines and Standard Operation Procedure
E 2 (8.4). The fish in stock were fed one to three times per day with 48 hours brine shrimp. The
feeding was stopped 24 hours prior to test initiation. The animals were not fed during the test. A
representative sample of test fish was measured at the end of the test and showed the length of the
animals to be =3 cm. Another representative sample of test fish was weighed before the start of the
test and showed that the biomass loading was

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

approx 12 °dH

Test temperature:

24.1 - 25.4 °C

pH:

8.1 - 7.7

Dissolved oxygen:

8.3 - 5.2 mg/L

Nominal and measured concentrations:

Nominal concnetrations: 0.06 - 0.125 - 0.25 - 0.5 and 1 mg/L

Details on test conditions:

As test vessels, 5 L glass aquariums containing 3 L of test solution were used. They were covered with
glass plates during the test.

The test was carried out in a temperature-controlled room with a light regime of 16 hours of ambient
light per day, provided by fluorescent tubes.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Details on results:

LC50: 0.3904 mg/L with 95% CL (0.3250-0.4689 mg/L)

Sublethal observations / clinical signs:

Survival and sub-lethal effects to fish

Conc. (mg/L)

No. of exposed fish

0 hours

4 hours

24 hours

48 hours

72 hours

96 hours

No. dead

Effects

No. dead

Effects

No. dead

Effects

No. dead

Effects

No. dead

Effects

No. dead

Effects

Control

7

7 AN

7 AN

7 AN

7 AN

7 AN

7 AN

0.06

7

7 AN

7 AN

7 AN

7 AN

7 AN

7 AN

0.125

7

7 AN

7 AN

7 AN

7 AN

7 AN

7 AN

0.25

7

7 AN

7 AN

7 AN

6AN 1*

5AN 2*

5AN 2*

0.5

7

7 AN

7 AN

4

3*

5

^2*

6

1*

6

1*

1.0

7

7 AN

4

1AN 1R 1E

7

-

-

-

-

-

-

R= lying on the bottom

* = contracted caudal fin / reduced activity

AN = appearing normal

E= erratic swimming

Validity criteria fulfilled:

not specified

Conclusions:

Due to lack of certificate of analysis there is no way of knowing what the substance actually is, and whether it contains impurities or is a mixture of different compounds. Therefore no reliable conclusion can be drawn at this moment.

Executive summary:

The acute toxicity of test item to the fresh water fish Danio rerio (zebra fish) was tested under static

conditions in accordance with OECD and EEC guidelines for testing of chemicals.

The fish were exposed to the following nominal test concentrations: 0.06, 0.125, 0.25, 0.5 and 1 mgll.

The LCs0 (96h) was calculated to be 0.3904 mgll with 95% confidence limits of 0.3250 and

0.4689 mgll

The highest concentration causing no mortality after 96 h amounted to 0.25 mg/L whereas 0.5 mg/L

was the lowest tested concentration (LOEC, 96 h) at which lethality was observed as compared to the

control. The LC100 was 1 mg/L, which was observed after 24 hours.

After 48 hours at 0.25 mgll reduced activity and contraction of the caudal fin was observed for one

fish. After 72 and 96 hours this effect was also observed for one other fish. Based on these

observations the NOEC is 0.125 mg/L.

At the concentrations where lethality was established, sublethal effects, such as reduced activity,

deviations in the behavior or the appearance of the fish, were observed. For ethical reasons suffering

fish in the test with 1 mg/L test substance were euthanised.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

96 hour

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

No analysis

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

analysis was not conducted

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No sampling

Details on test solutions:

The test substance was insoluble in water, but dispersable. Test solutions were produced by making "dispersions" of the test substance in deionised water. Stock solutions of 1g/l were prepaired in 200 ml of deionised water stirred at room temperature. This was not sufficient to dissolve so
ultrsonication was used to achieve a homogenious solution. This solution was then made up to one liter. Test solutions were made by serial dilution of this stock using dutch standard water to achieve test concentrations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Fish obtained from established local retailer. Batch 16-3-1995. Fish recieved quarantine before being used in test. Kept according to laboratorium
standard operating procedures. Size range : 2.1-2.4 cm Weight 0.08g-0.16g. Fish were not fed 24 hours prior to test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Not measured in actual test reported as approximately 11.7 dh

Test temperature:

Measured ranged reported to have been from 21-23 degrees centigrade. Not specifically noted in report.

pH:

7.9-8.1. pH did not vary more than one unit (in all test concentrations) throughout the test.

Dissolved oxygen:

Was maintained at >60% of the air saturation value.

Salinity:

Not Stated

Nominal and measured concentrations:

Preliminary-0.1-1.0-10-100mg/l Nominals Only
Definative-0.1-0.18-0.32-0.58-1.05 mg/l Nominals Only

Details on test conditions:

Test was carried out under temperature controlled conditions. Control temperature was continually recorded. 16 hours ambient light per day. Test vessels were continually aerated. Fish and aquaria were randomly placed.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%

Details on results:

NOEC= 0.1mg/l
100% mortality after 30 hours = 0.32mg/l

Reported statistics and error estimates:

LC 50 was calculated with AkZO SKBT version 1.0 using trimmed Spearman-Karber and binomial test method.

Sublethal observations / clinical signs:

Mortality in definative Test                                          

Test Duration Hours	0	2	4	7	23	24	30	47	48	55	71	72	78	95	96
Mg Test Substance
control	7	7	7	7	7	7	7	7	7	7	7	7	7	7	7
0.1	7	7	7	7	7	7	7	7	7	7	7	7	7	7	7
0.18	7	7	7	7	7	7	7	6	6	6	6	6	6	6	6
0.32	7	7	7 a	7a	1a	1a	0
0.58	7	7a	6a	2a	0
1.05	7	7a	1a	0

a= reduced activity.

Validity criteria fulfilled:

yes

Remarks:

Criteria Filled With Exception of Analysis

Conclusions:

Test generally well conducted however no analysis was condutcted. Concentrations are therefore all nominals and not completely reliable. Factors
such as solubility and possible adhesion of test chemical to glasware dont appear to have been considered in enough detail to give complete
confidence in the nominals. Water hardness not specifically recorded during test only an approximation could be given. Temperature recorded but not displayed in results approximate range given.

Executive summary:

The LC50 96H was calculated to be 0.22mgll with 95% confidence limits of 0.19 and 0.26mg/l. The highest causing no mortality (no observed effec concentration, NOEC) after 96 hours was 0.1mg/l. 100% mortality was achieved at 30hours at 0.32mg/l. At 0.32,0.58 and 1.05 mg/l all fish showed reduced activity prior to death.

Description of key information

Amides, C18-unsatd., N-[3-(dimethylamino)propyl] were found to be very toxic to fish with EC50 of 0.94 mg/L, expressed as nominal concentrations.

It is assessed from the methods of manufacture the starting materials and the similarity of the end product compositions that EC272-047-7 is sufficiently similar in all respects to EC800-353-8 that it is a sensible approach to use a read-across approach. A comparison of the two substances and a read-across justification can be found in section 13 of this dataset.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.94 mg/L

Additional information

The acute toxicity of Amides, C18-unsatd., N-[3-(dimethylamino)propyl] to the fresh water fish Danio rerio (zebra fish) was tested under semi static conditions,with refreshment after 48 hours in natural water for 96 hours in accordance with the OECD and EEC guidelines for testing of chemicals, with minor modifications. The test wasperformed in compliance with Good Laboratory Practice (GLP).

 

The definitive test was performed with the following nominal test concentrations including a control: 0.2, 0.45, 1.0, 2.2 and 4.4 mg/L.

 The NOEC for the test substance was determined as 0.45 mg/L. The LC₅₀calculated using the Spearman Karber Graphical Method was a 0.94 mg/L.

 

Chemical analysis of the initial concentrations of the test chemical was carried out at all concentrations and in the stock solutions. Recovery of the test chemical after 48 hours was not possible due to inherent test chemical properties. Endpoints were therefore based on nominal values confirmed by the initial analytical recoveries of >90% of the nominal concentrations, in line with the bulk approach.

 

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