Allylamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2013 to 28 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with international guidelines (OECD 202), and in compliance with the applicable GLP Regulations. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Completely filled and sealed Wheaton vials (35 mL) were used for samples. At the start of the definitive test, two samples were taken from the freshly-prepared control and test media in one set of destructive vials. After 48 hours, two further samples were taken for analysis from the remaining destructive vials for each group.
On each occasion, one of the samples was analysed and the other was left sealed stored at ambient temperature in case further analysis was required.

Vehicle:

no

Details on test solutions:

The method of preparation used during the definitive test was based on the results of a formulation trial and two range finding tests. Due to the volatile nature of the test substance, the study was conducted in completely filled and sealed glass vials (nominal capacity, 125 mL). The test substance also resulted in an increase in the pH of the dilution medium, so the definitive test was conducted using test media prepared with and without pH adjustment.
At each concentration, an aliquot of the test substance (13.1, 28.8, 63.5, 140 or 310 µL) was added to dilution medium (1 L) in a volumetric flask (1 L); the contents of the flask were shaken, before being used to completely fill six vials (leaving no headspace) to provide test media with nominal concentrations at 10.0, 22.0, 48.4, 106.5 or 234.3 mg/L as allylamine. No adjustment for pH was made to this batch of test media.
At each of the three highest concentrations (48.4, 106.5 and 234.3 mg/L as allylamine), a second batch of test media was prepared by adding an aliquot of test substance (63.5, 140 or 310 µL) to dilution medium (700 mL) in a volumetric flask (1 L); the pH of this media was adjusted to within 8.5 ± 0.2 using 0.1M HCl. The media were made up to volume, shaken and the pH re-measured before being used to completely fill six vials at each level, to provide the pH-adjusted test media.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the National Institute for Applied Chemical Research (IRCHA), France.
Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

See Table 2.

Test temperature:

See Table 2.

pH:

See Table 2.

Dissolved oxygen:

See Table 2.

Salinity:

See Table 2.

Nominal and measured concentrations:

Nominal Concentrations: 0, 10, 22, 48.4, 106.5, 234.3 mg/L
Mean Measured Concentrations: 0, 9.61, 19.2, 43.4 (42.4), 78.7 (117), 255 (256) - Brackets denote pH adjusted media.
Further details presented in Table 1.

Reference substance (positive control):

yes

Duration:

24 h

Dose descriptor:

EC50

Remarks:

non-pH adjusted

Effect conc.:

28.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL [19.2, 43.3]

Duration:

24 h

Dose descriptor:

EC50

Remarks:

pH adjusted

Effect conc.:

194 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL [176, 256]

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

non-pH adjusted

Effect conc.:

28.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL [19.2, 43.4]

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

pH adjusted

Effect conc.:

173 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL [117, 256]

Duration:

48 h

Dose descriptor:

LOEC

Remarks:

non-pH adjusted

Effect conc.:

43.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Remarks:

pH adjusted

Effect conc.:

256 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Remarks:

non-pH adjusted

Effect conc.:

19.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Remarks:

pH adjusted

Effect conc.:

117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

At all concentrations, the intended exposure levels of allylamine were adequately achieved (between 63 and 115% of their nominal values) and maintained during the test (between 72 and 136% of their starting values). The overall mean measured levels of allylamine were 9.61, 19.2, 43.4, 78.7 and 255 mg/L for samples non-adjusted for pH, and 42.4, 117 and 256 mg/L for samples adjusted for pH.. These values have been used in the calculation of test results.
Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours. After 24 hours in non pH-adjusted media, there was 100% immobility at measured concentrations of 43.4, 78.7 and 255 mg/L. In pH adjusted media, there was 85% immobilisation at 256 mg/L, which increased to 100% at 48 hours. This implies that the pH of the test substance was responsible for observed immobilisation at nominal concentrations of 48.4 and 106.5 mg/L, with the test substance itself having a measurable impact at the highest test concentration only.

Results with reference substance (positive control):

The sensitivity of juvenile Daphnia cultured in this laboratory is periodically assessed using the reference substance potassium dichromate. The results for the most recent test performed prior to this study indicated that its 48-hour EC50 to Daphnia magna was 0.934 mg/L; this was considered comparable with data previously obtained in this laboratory.

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application (version 1.5), SAS 9.1.3 (SAS Institute, 2002). Test results were expressed in terms of the nominal and mean measured concentrations with and without pH adjustment.
The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Measured Concentrations

Concentration	Measured Concentrations (mg/L)					Overall Mean *
(mg/L)	0 hours	% Nominal	48 hours	% Nominal	%ti	(mg/L)
Control	nd	-	nd	-	-	nd
10.0	8.24	82	11.2	112	136	9.61
22.0	17.7	80	20.8	95	118	19.2
48.4	40.8	84	46.1	95	113	43.4
48.4 ^	47.9	99	37.6	78	78	42.4
106.5	92.7	87	66.8	63	72	78.7
106.5 ^	112	105	122	115	109	117
234.3	264	113	246	105	93	255
234.3 ^	260	111	253	108	97	256

nd - None detected (less than the limit of detection of 0.4 mg/L)

% ti - Mean measured concentration after 48 hours expressed as a percentage of the mean starting concentration

* Geometric mean

^ Media with pH adjustment

Table 2. Environmental Parameters

Concentration (mg/L)		Temperature (°C)		pH		Dissolved Oxygen (% ASV)
Nominal	Measured	0 h	48 h	0 h	48 h	0 h	48 h
Control	nd	20.6	21.1	7.83	7.65	99	97
10.0	9.61	20.4	21.0	8.91	8.56	99	97
22.0	19.2	20.3	20.9	9.29	9.21	99	98
48.4	43.4	20.3	20.7	9.77	9.43	99	96
48.4 ^	42.4	20.3	20.7	8.43	8.35	99	96
106.5	78.7	20.3	20.7	10.17	10.01	99	98
106.5 ^	117	20.3	20.7	8.70	8.59	99	95
234.3	255	20.4	20.7	10.56	10.28	100	99
234.3 ^	256	20.3	20.8	8.68	8.62	100	96

^ - Media with pH adjustment.

nd - None detected (less than the limit of detection of 0.4 mg/L).

ASV - Air saturation value (100 % ASV = 9.09 mgO2/L at 20 °C)

The total hardness and alkalinity of the batch of Elendt M4 medium used were 236 and 49 mg/L as CaCO3, respectively.

Continuous monitoring of an additional vessel containing dilution medium = 19.8 to 20.8°C.

Table 3. Cumulative Immobilisation

Concentration (mg/L)		Cumulative Numbers of Immobile Daphnia
Nominal	Mean Measured	24 hours						48 hours
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	nd	0	0	0	0	0	0	0	0	0	0	0	0
10.0	9.61	0	0	0	0	0	0	0	0	0	0	0	0
22.0	19.2	0	0	0	0	0	0	0	0	0	0	0	0
48.4	43.4	5	5	5	5	20	100	5	5	5	5	20	100
48.4 ^	42.4	0	0	0	0	0	0	0	0	0	0	0	0
106.5	78.7	5	5	5	5	20	100	5	5	5	5	20	100
106.5 ^	117	0	0	0	0	0	0	0	0	0	0	0	0
234.2	255	5	5	5	5	20	100	5	5	5	5	20	100
234.3 ^	256	5	5	3	4	17	85	5	5	5	5	20	100

nd - None detected (less than the limit of detection of 0.4 mg/L)

Rn - Replicate number.

^ - Media with pH adjustment.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations, the 48-hour EC50 of allylamine for the immobilisation of Daphnia magna was 28.9 mg/L when performed without pH adjustment and 173 mg/L following pH adjustment of the media.
The ‘no-observed effect concentration’ (NOEC) was 19.2 mg/L without pH adjustment and 117 mg/L with pH adjustment.
These results suggested that the pH of the medium was primarily responsible for the immobilisation observed at nominal 48.4 or 106.5 mg/L as no immobilisation was observed in vessels containing media that had been pH adjusted. Immobilisation at nominal 234.3 mg/L is considered to be related to the test substance as 100% immobilisation was observed irrespective of the pH of the media.

Executive summary:

The acute toxicity of allylamine to Daphnia magna was assessed under static exposure conditions. Due to the volatile nature of the test material the test was conducted in sealed vessels with no headspace.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia Acute Immobilisation Test” (2004).

Groups of twenty Daphnia, less than 24 hours old, were exposed for 48 hours to allylamine, at nominal concentrations of 10.0, 22.0, 48.4, 107 and 234 mg/L. At each concentration, the test medium was prepared by the direct addition of the test substance to dilution medium (Elendt M4 medium). At the three highest concentrations, where the pH of the test media increased to above 9.5, additional batches of test media were prepared in which the pH was adjusted to ca. 8.5 before use.

At all concentrations, the intended exposure levels of allylamine were adequately achieved (between 63 and 115% of their nominal values) and maintained during the test (between 72 and 136% of their starting values). The overall mean measured levels of allylamine were 9.61, 19.2, 43.4, 78.7 and 255 mg/L for samples non-pH adjusted media, and 42.4, 117 and 256 mg/L for samples adjusted for pH. Both data sets have been used in the calculation of test results.

Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours. After 48 hours in non pH-adjusted media, there was 100% immobility at measured concentrations of 43.4, 78.7 and 255 mg/L. In pH adjusted media, 100% immobilisation was only observed at 256 mg/L at 48 hours. This implies that it was the pH of the test media that was primarily responsible for the immobilisation observed and that the test substance itself only had a toxic effect at the highest test concentration.

Based on these findings, the following study end-points values have been determined:

	Mean Measured Concentration (mg/L)
Parameter	Non-pH adjusted	pH adjusted
24 -hour EC50	28.9 [19.2, 43.4]	194 [176, 256]
48 -hour EC50	28.9 [19.2, 43.4]	173 [117, 256]
LOEC	43.4	256
NOEC	19.2	117

NOEC - No observed effect concentration. LOEC - Lowest observed effect concentration. [ ] - 95% confidence intervals.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was not conducted according to GLP, analytical monitoring of test concentrations was not conducted, and no water quality parameters were presented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia cucullata

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

No data

Test temperature:

The study was conducted at 19 °C.

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

No data

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 28 - <= 29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No CL published

LC50 48-hour values to Daphnia cucullata

	a*	b*	mean
Allylamine	29	28	29

 * - Values from two seperate studies

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of allylamine to D. cucullata was 29 mg/L.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was not conducted according to GLP, no analytical monitoring of test concentrations was conducted and no water quality parameters are presented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

Standard culturing techniques were used, and animals used in the toxicity test were <24 hours old at test iniation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

No data.

Test temperature:

The study was conducted at 19 °C.

pH:

No data.

Dissolved oxygen:

No data.

Salinity:

No data.

Nominal and measured concentrations:

No data.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 30 - <= 48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No CL published

48-hour LC50 values (mg/L) to Daphnia magna

	Laboratory 1 (exp. 1)			Laboratory 2 (exp. 1)			Laboratory 2 (exp. 2)
	a	b	mean	a	b	mean	a	b	mean
LC50	32	30	31	42	43	43	48	46	47

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of allylamine to Daphnia magna was estimated as 30 mg/L.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was not conducted according to GLP, analytical monitoring of test concentrations was not conducted, and no water quality parameters were presented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia pulex

Details on test organisms:

D. pulex were cultured using standard methods. Neonates were <24 hours old at study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

No data

Test temperature:

The study was conducted at 19 °C

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

No data

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 32 - <= 36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No CL published

LC50 48 -hour values (mg/L) to Daphnia pulex

	a	b	mean
EC50	32	36	34

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of allylamine to D. pulex was 32 mg/L.

Description of key information

48h EC50 (Daphnia magna) = 28.9 mg/L; OECD 202; Allen (2013).

48h EC50 (Daphnia magna) = 30 mg/L; similar to OECD 202; Canton and Adema (1978).

48h EC50 (Daphnia pulex) = 32 mg/L; similar to OECD 202; Canton and Adema (1978).

48h EC50 (Daphnia cucullata) = 29 mg/L; similar to OECD 202; Canton and Adema (1978).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

28.9 mg/L

Additional information

A single key study (Allen, 2013) is submitted for acute toxicity to fish. The study was conducted in accordance with OECD 202, and was conducted in accordance with GLP. All relevant validity criteria were met in this study.