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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

72 hours

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was performed according to OECD guideline and GLP requirements. But due to the properties of the test substance, the concentrations were higly decreased from the start of the test and no substance was measured at the end of the test for the four lowest concentrations tested. Indeed, the test substance is poorly soluble in water and has a tendency to bind to negatively charged surfaces such as glass, suspended matter, algae, or other organic material. Therefore, standard guideline studies are considered inappropriate for cationic substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The test substance concentrations decreased more than 20% over the course of the study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The test substance concentrations decreased more than 20% over the course of the study

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All the concentrations were analysed at the begining of the test and in non-inoculated and inoculated series at the end of the test. By comparing the test item concentrations between these series, the stability of the test item and its potential absorption, adsorption or metabolization by algae was evaluated.
Dissolved O2 and pH were measured in the control and the highest concentration, in non inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.

Vehicle:

no

Details on test solutions:

Since the test substance is a multi-component mixture and is not soluble in dilution water, WAF were used. For the preliminary test, WAF were prepared at different concentrations, the day before the beginning of the test. Solutions were filtered through HVLP 0047 Millipore filter (0.45 µm) allowing to obtain a limpid solution. For the definitive test a stock solution of the test item diluted in water was prepared one day before the beginning of the test (time 0) by mixing 10 mg of test item in one liter of dilution water. Indeed, the effects on the growth of algae have been observed at very low concentrations in the preliminary test. Therefore, it was not possible to use WAF in the definitive test at the tested concentrations.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- unicellular green fresh-water algae Pseudokirchneriella subcapitata, CCAP 278/4 stock obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK)

ACCLIMATION
- Culturing media and conditions:
- 2 flasks containing 100 mL of axenic stock culture of algae incubated at 23 °C (+ or - 1 °C)
- Aeration: continuous (air filtered at 0.22 µm)
- Lighting: photoperiod: 16 hours of illumination, 8 hours of darkness
- 3 to 5 days bofore the beginning of the study, 2 pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures. The cell density of the pre-culture was about 3.51 10^6cells/mL for the preliminary test and about 3.4 10^6 cells/mL for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

maintained at 23.5ºC ± 1ºC

pH:

T0 = 7.96;
T72h = 7.89 to 8.49

Dissolved oxygen:

T0 = 9.0 to 9.1 mg O2/L
T72h = 8.7 to 9.5 mg O2/L

Salinity:

no data

Nominal and measured concentrations:

Nominal concentrations in the definitive test: 0-20-40-90-210-450-1000 µg/L
For the measured concentrations, see the table below.

Details on test conditions:

The final concentrations of the test substance were not maintained within the designated limit of 80 % of the initial concentrations in non-inoculated flasks. Thus, the test item concentrations were not maintained throughout the duration of the test.
Therefore, calculations of effective concentrations EC50, EC10 and NOEC were performed using the geometric averages values between initial and final concentrations of the test substance.

Reference substance (positive control):

yes

Remarks:

potassium dichromate K2Cr2O7, volumetric standard quality

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Dose descriptor:

EC10

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Dose descriptor:

EC10

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless, became not cloudy over the period of the test.
- All algae examined microscopically at test termination appeared normal.
- Since the initial measured concentrations in non-inoculated flasks were not found to be equivalent (>80%) to the nominal concentrations and the final test substance concentrations measured in non-inoculated flasks were not found to be equivalent (>80%) to the initial not inoculated ones, calculations of effective concentrations EC50, EC10 and NOEC were performed using the geometric average values between initial and final concentrations of the test substance.

Results with reference substance (positive control):

Most recents values of ErC50 and EbC50 obtained on July 08 2004 were respectively 0.93 and 0.34 mg/L. For information, ISO 8692 reports the following results for an inter-laboratory exercise on sodium dichromate: ErC50: 0.6 to 1.03 mg/L and Eb50: 0.2 to 0.75 mg/L.

Reported statistics and error estimates:

The relationship between the toxicity (inhibitionof growth) and the test item concentration does not fit a linear model. Consequently, the average percentage inhibition are plotted against concentrations using the non-linear Probit/log model.

Test item concentrations during the definitive test

	Concentrations of test item during the test
Nominal concentration (µg/L)	Measured in non inoculated solutions			Extrapolated (µg/L)*
	Initial (µg/L)	Final (µg/L)	Final/Initial (%)
0	0	0	-	-
20	5.5	1.5**(1.5**)	27	3
40	24	1.5** (1.5**)	6.2	6
90	53	5.5*** (1.5**)	10.4	17
210	110	21 (1.5**)	19	48
450	220	63	28.6	118
1000	420	290	69	349
LD = 3 µg/L
LQ = 11 µg/L
*concentrations calculated with geometric average of final and initial concentrations.
** half the value of the detection limit was used because the value was <LD
*** half the value of the quantification limit was used because the value was <LQ.
Within parenthesis: concentrations of test substance measured in inoculated series (with algae)

Percentage of inhibition of growth rate during the definitive test

Nominal concentration (µg/L)	Inhibition of growth rate (%)
0	0
20	9.89
40	87.82
90	110.89
210	142.71
450	181.08
1000	181.08

Validity criteria fulfilled:

no

Remarks:

see details in conclusions

Conclusions:

Increase in cell density in control > 16 (R = 66)
But concentrations of the test item not maintained within the designated limit of 80% of the initial concentrations.

The determination of the growth inhibition (chronic toxicity) of the freshwater algae Pseudokirchneriella subcapitata exposed to the test substance for a duration of 72 h was performed following the method C3 described in Directive 92/69/EEC of the European Commission and in the guideline 201 of the OECD.
The concentration and loading of test item causing a 50% reduction in biomass (EbC50 and EbL50) or in growth rate (ErC50 and ErL50) were determined.

The ErC50-72h obtained was 0.005 mg/l. This very low value may be related to a true algicidal effect of the test substance.

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to Amides, rape-oil, N-[3-(dimethylamino)propyl] for a duration of 72 hours was performed following the method C3 described in Directive 92/69/EEC of the European Commission and in the guideline 201 of the OECD. The validity criterion of the study related to the growth of algae was respected. But the concentrations were not maintained within the designated limit of 80% of the initial concentrations and were decreased from the start of the test. Therefore, results are expressed using the geometric average values between initial and final concentrations of the test substance. ErC50 -72h and ErC10 -72h were estimated to be 0.005 and 0.002 mg/L, respectively.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

72 hours

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Bulk approach applied, Natural water used

Principles of method if other than guideline:

The bulk approach was applied. The emphasis of the chemical analysis was therefore the accurate determination of the bulk concentration of the test chemical added at the start of the test.

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):
N/A

Analytical monitoring:

yes

Details on sampling:

Samples of 10 mL were taken at the start and end of the test at all concentrations (including parallel), in the stock solution and in the control.

Vehicle:

no

Details on test solutions:

A stock solution of 101 mg/L of the test substance was prepared by accurately weighing 0.0101 g of test substance, on an analytical balance. The test chemical was then added to approximately 60 mL of the test media. This solution was homogenised for one minute a homogeneous stock solution was achieved. The volume was made up to 100 mL with the test medium. The stock was kept stirring while in use to generate the test concentrations. Stock pH was 8.3. No pH adjustment was required. This stock was subsequently diluted a further 10 times to allow more accurate pipetting the test concentrations

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.

The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1x10 4 cells/mL in each of the test vessels.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

At the end of the test five random samples were microscopically checked for purity of the algal culture.

Hardness:

6.71ºdH

Test temperature:

The temperature varied from 21.65 to 22.1 °C during the test.

pH:

Maximum increase of 0.3 pH units in the control replicates and in some of the test concentrations.

Dissolved oxygen:

N/A

Salinity:

N/A

Nominal and measured concentrations:

Nominal concentrations of 0.015, 0.045, 0.13, 0.41 and 1.2 mg/L were tested. For the highest 2 concentrations a statistically significant effect to growth rate in comparison to the control was detected.
Measured concentrations were > 80% of the nominal concentrations in the stock and in the highest 2 concentrations. The analytical data at the end of the study in the test vessels themselves as well as in the parallel replicates fall below the quantification limit of the analytical method in most cases likely due to the adsorbing properties of the test substance. The collected data clearly demonstrates the test substance to be strongly sorbing and therefore the end of test analytics are not truly representative of the realistic exposure as the test substance was likely to bind directly to the test organism as is well documented for surface active substances. The endpoints will therefore be based on measured initial values as indicated in the test guideline as acceptable for algae studies with adsorbing substances at low concentrations.

Details on test conditions:

Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with natural surface water. Natural water was frozen prior to use to reduce microbial loading but was not sterilized. Sterilized Erlenmeyer flasks were then filled with test medium up to the appropriate volume using a calibrated dispenser pump. Adequate amounts of test substance were then added from the stock solution (see below) to achieve the desired test concentrations in a total volume of 40 mL. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control and a parallel replicate of every concentration without algae were included. The absorbance in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Test medium (at each relevant concentration) was used as a blank in the spectrophotometer.

The light intensity was measured at the beginning and at the end of the test using a light intensity meter: the light intensity was 99.8 and 100.1 µmol.m-2.s-1 at the beginning and end of the test respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.103 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.96 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Due to 41% inhibition being reached during the test, an extrapolation to 50% can be considered acceptable due to its proximity to the highest concentration: ErC50 (72h)extrapolated value was calculated as 1.17 mg/L.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity. The EC50 value of the reference compound was in the range of 0.25-2.0 mg/L.

Reported statistics and error estimates:

During the definitive study all of the desired endpoints could be calculated. Dose response data with confidence limits were calculated for the biomass endpoint but could not be calculated for the rate endpoint due to the highest concentration resulting in approximately 50% inhibition. This does not affect the reliability for the key endpoints but extrapolation above the ErC50 is not considered reliable.
ErC10 and ErC50 values were calculated by a maximum likelihood probit plot. NOEC and LOEC were also determined using a Dunnetts test. Normal distribution and equality of variance were tested by the Shapiro-Wilks and the Bartlett’s tests. The data was indicated to be normal and equal in variance.

Table:1a:Measured concentrations of the test samples (mg/L)

sample	0h	% of nominal		72h	72h parallel
Control	< LOQ	-		< LOQ	< LOQ
0.015 mg/L	0.010	77.2		< LOQ	< LOQ
0.045 mg/L	0.035	79.4		< LOQ	< LOQ
0.13 mg/L	0.103	77.1		< LOQ	< LOQ
0.41 mg/L	0.316	80.0		0.001	< LOQ
1.2 mg/L	0.960	99.2		0.001	< LOQ
stock	100.2

LOQ-limit of quantification

Table1b:Validity criteria of the analytical method

Parameter	Limit	Determined
Squared regression coefficient (R^2)	= 0.98	= 0.9999
Accuracy	80 % - 120 %	82.3 – 101.4
Repeatability (CV (%)) at lowest standard	= 20	= 0.6
Repeatability (CV (%)) at Highest standard	= 20	= 0.4
LOQ (µg/L)	7.5	0.45
System stability (% of nominal)	= 10	= 3.9
Recovery from medium (%)	= 70 and = 120	92.2

Table 2 Raw data

Nominal Concentration	ABSORBANCE   Time (hours)				AUC	Inhibition %	SGR	Inhibition %
(mg/L)	0	24	48	72
Control	0.001	0.009	0.241	1.005	18.00		0.1
Control	0.001	0.001	0.246	0.978	17.604		0.109
Control	0.001	0.013	0.257	0.994	18.348		0.099
Control	0.001	0.010	0.240	0.992	17.844		0.099
Control	0.001	0.006	0.244	1.005	18.0		0.102
Control	0.001	0.015	0.249	0.972	17.94	0	0.098	0
Mean
0.015	0.001	0.019	0.252	0.999	18.432		0.097
0.015	0.001	0.012	0.272	1.025	19.056		0.100
0.015	0.001	0.006	0.249	0.967	17.664		0.101
Mean					18.384	-2.4	0.099	1.0
0.045	0.001	0.008	0.227	0.982	17.364		0.100
0.045	0.001	0.009	0.248	0.958	17.604		0.100
0.045	0.001	0.012	0.224	0.931	16.776		0.098
Mean					17.248	3.9	0.099	1.0
0.13	0.001	0.006	0.227	0.364	16.836		0.101
0.13	0.001	0.001	0.218	0.401	16.176		0.108
0.13	0.001	0.004	0.204	0.354	15.840		0.102
Mean					16.284	9.3	0.102	-2.3
0.41	0.001	0.001	0.080	0.364	6.252		0.092
0.41	0.001	0.001	0.067	0.401	6.384		0.092
0.41	0.001	0.002	0.079	0.354	6.132		0.088
					6.256	65.2	0.091	9.2
Mean
1.2	0.001	0.030	0.050	0.113	3.216		0.061
1.2	0.001	0.031	0.039	0.098	2.796		0.058
1.2	0.001	0.022	0.042	0.080	2.436		0.057
					2.816	84.3	0.059	40.9
Mean

AUC= Area under curve (Biomass)

SGR = Specific Growth Rate (Rate)

Validity criteria fulfilled:

no

Remarks:

see details in the conclusion section

Conclusions:

The following quality criteria have been met in the present study:

• The cell density of the controls increased at least a factor 16 within 72 hours.
• The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%.
• The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L.


But the validity criteria of the analytical method were met.


The following criteria were not met:

• The mean coefficient of variation for section-by-section specific growth rates in the control did exceeded 35 %. This was greatly influenced by the presence of suspended matter and the inaccuracies associated with correcting for this before algae growth has taken place. Due to good algae growth and very little variation in the end absorbance between controls at the end of the test (after algae growth) the test system was still considered valid. Due to the presence of suspended matter this variation is a direct result of the test medium used not variation in growth.
• The test substance concentration decreased below 80% of the nominal and initial concentrations during the test. This is not uncommon with adsorbing substances and is not in this case indicative of reduced exposure. Surfactant based substances are known to bind to algae cells actually stimulating complete exposure. This is however not measurable analytically. This difficulty is acknowledged in the OECD test guideline. Use of the measured initial concentration is therefore considered acceptable for this test substance.

Executive summary:

An algal growth inhibition test with Amides rape oil N-[dimethyl amino propyl] was conducted in accordance with OECD Test Guideline 201, and in compliance with the OECD principles of Good Laboratory Practice. Specific modifications to the guideline were that natural surface water was used for testing which was supplement with the recommended nutrients from the OECD algal medium at 50% concentration and NaHCO3 increased to 75 mg/L. The bulk approach was applied to the test methodology allowing the use of total aquatic concentrations for calculation of endpoints.  The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L. The 72 hour ErC10 was calculated as 0.32 mg/L and the ErC50 > 0.96 mg/L (extrapolated value = 1.17 mg/L), expressed as measured initial values.

Description of key information

Amides, C18-unsatd., N-[3-(dimethylamino)propyl] were found to be toxic to green algae with an extrapolated ErC50 of 1.17 mg/L and an ErC10 of 0.32 mg/L, expressed as measured 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

EC50 for freshwater algae:

1.17 mg/L

EC10 or NOEC for freshwater algae:

0.32 mg/L

Additional information

An algal growth inhibition test with Amides, C18-unsatd., N-[3-(dimethylamino)propyl] was conducted in accordance with OECD Test Guideline 201, and in compliance with the OECD principles of Good Laboratory Practice (Kean, 2013). Specific modifications to the guideline were that natural surface water was used for testing which was supplement with the recommended nutrients from the OECD algal medium at 50% concentration and NaHCO3 increased to 75 mg/L. The bulk approach was applied to the test methodology allowing the use of total aquatic concentrations for calculation of endpoints. The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L. The 72 hour ErC10 was calculated as 0.32 mg/L and the ErC50 > 0.96 mg/L (extrapolated value = 1.17 mg/L), expressed as measured initial values.