2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2003 to 07 November 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed to GLP and in line with the standardised guidelines OECD TG 201 with some minor deficiencies present within the methodology and reporting. A drift of greater than 1.5 pH units was noted during the course of the test (contrary to the guideline); though this is not considered to impact the accuracy and reliability of the results. Although microscopic evaluations were not available to determine the effect of the pH on the integrity and morphology of the cells, the growth rates demonstrated a lack of effect by the pH increase. The control demonstrates exponential growth throughout the entire study (as with all the test vessels) and the extreme pH did not exert any toxic effects. No effects of the pH were noted in the test substance treated vessels (which had the highest pH drifts) as exponential growth was noted in all vessels and a dose related response was noted at the higher dosing levels, demonstrating that the test substance did not act as a substrate for respiration. These deviations were not considered to affect the accuracy of the overall conclusions and the study is suitable for classification and labelling purposes but does not allow a detailed assessment of the possible effects of the substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

no microscopic evaluations performed, pH of control drifted more than 1.5 units

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations tested.
- Sampling method: Samples for analysis were taken at 0 hours from the excess test solutions and at 72 hours from one of the exposure replicates. At 72 hours, samples were centrifuged for 30 minutes at 40000 G prior to sample treatment to remove algal cells.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 2000 mL volume of the highest nominal concentration test solution (100 mg/L) was prepared by the direct addition of the test material to sterile culture medium. This stock solution was given 10 minutes ultrasonic treatment and stirred for 10 minutes. The lower test concentrations were prepared, using sterile culture medium, by the addition of aliquots of the nominal 100 mg/L test solution to final volumes of 1000 mL. After at least 15 minutes stirring the resultant 0.78 to 13 mg/L solutions were clear and colourless and the 25 to 100 mg/L solutions were clear, very pale yellow solutions increasing in intensity with concentration. The control consisted of culture medium only. Using aseptic techniques, 100 mL volumes of the appropriate test solutions were dispensed to each test and blank vessel, with the remainder of the test solutions being used for physical and chemical analyses.
- Controls: Dilution water/medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Selenastrum capricornutum ATCC 22662
- Source: Laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Axenic conditions

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture was grown in the medium, and under the environmental conditions of the test.
- Any deformed or abnormal cells observed: Not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.9 to 24.0 °C (24 ± 2 °C)

pH:

Start: 7.37 to 7.45
End: 9.03 to 10.25

Nominal and measured concentrations:

0.78, 1.6, 3.1, 6.3, 13, 25, 50 and 100 mg/L nominal test material in solution

0.74, 1.6, 3.1, 6.3, 12, 25, 53 and 100 mg/L measured test concentration (mean of triplicate assays)

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): Closed (polyurethane foam bungs)
- Test vessel, type, material, size, headspace, fill volume: borosilicate glass conical flasks of 250 mL nominal capacity.
- Aeration: The vessels were shaken throughout the study
- Initial cell density: Each replicate test vessel was inoculated with 0.75 mL of the inoculum culture to give a nominal cell density of 1.00 x 10^4 cells/mL
- Control end cells density: 290 x 10^4 cells/mL (average of six replicates) (please refer to table 3 under the field “Any other information on results incl. tables”).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (U.S. EPA medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The medium was prepared using the steps presented under the field “Any other information on materials and methods incl. tables”.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous "cool-white" illumination
- Light intensity and quality: The light intensity was measured once during the test. 7100 lux (by cosine receptor); 88.4 µ Einsteins m^-2 s^-1

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Electronic particle counter. Three 100 mL volumes of Coulter electrolyte, inoculated in the same manner as the test vessels, had a mean measured cell density of 1.03 x 10^4 cells/mL. This was used for growth calculations.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- pH shift: At the start of the test the pH ranged from 7.37 to 7.45 and at the end of the test the range was 9.03 to 10.25. A maximum increase of 2.83 pH units was observed over the test duration. This pH shift was considered to be a function of the very high growth factors observed (mean culture medium control 72 hours cell density = 282 x 0 hour initial inoculum density). The pH shift occurred despite a high orbital shaking rate of 160 rpm to improve mass transfer of carbon dioxide into the test solution.

Reported statistics and error estimates:

- Areas under the growth curve:
The area under the growth curve (0 to 72 hours) was calculated for each replicate culture, according to the following:

Area = ((N0+N1)-2N0/2) x t1 + ((N1+N2)-2N0/2) x (t2 - t1) + ((Nn-1 + Nn) – 2N0/2) x (tn – tn-1)

Where:
N0 = Cell density at start of test
N1 = Cell density at t1
Nn = Cell density at tn
t1 = Time (days) of first measurement after start of test
t2 = Time (days) of second measurement after start of test
tn = Time (days) of nth measurement after start of test

These areas were examined by one-way analysis of variance, and Dunnett's procedure (Ref 5) was used to identify significant differences (P=0.05) from the culture medium control. As less than 50% inhibition was observed over the range of concentrations tested, the ErC5O is considered to be greater than the highest concentration tested for each timepoint.

- Growth rates:
The growth rate (0 to 72 hours) was calculated for each replicate culture, according to the following:

Growth rate = Logn(N2/N1)/t

Where:
N1 = Cell density at start of test
N2 = Cell density at end of test
t = Time interval (days)

Because the growth rate at each concentration was relatively constant throughout the test period, it was considered not necessary to analyse growth rates over shorter intervals.

Table 2: Analytical results

Nominal conc of test material (mg/L)	Measured concentration of test material (mg/L)				Mean measured concentration over test duration^a(mg/L)	Mean measured concentration as percentage of nominal concentration	Percentage loss in concentration over test duration^b
	0 hours	% of nominal	72 hours	% of nominal
Culture medium control	<0.012	-	<0.012	-	<0.012	-	-
0.78	0.75	96	0.73	94	0.74	95	3
1.6	1.6^c	100	1.6^c	100	1.6	100	0
3.1	3.1	100	3.1	100	3.1	100	0
6.3	6.3	100	6.3	100	6.3	100	0
13	12	92	11	85	12	92	8
25	25	100	25	100	25	100	0
50	53	106	52	104	53	106	2
100	100	100	100	100	100	100	0

^aCalculated using the arithmetic mean of the 0 and 72 hour results

^b100 -[(final analysed concentration/concentration at 0 hours) x 100]

^c Mean of triplicate analyses

The Limit of Quantitation was 0.012 mg/L.

Table 3: Algal cell densities

Nominal concentration of test material (mg/L)	Replicate	Algal cell density (x 10^4cells/mL)
		24 hours	48 hours	72 hours
Culture medium control	A	7.26	48.4	256
	B	7.26	58.6	322
	C	6.61	55.0	300
	D	6.45	51.2	282
	E	6.52	54.4	306
	F	6.16	49.3	273
	Mean	6.71	52.8	290
0.78	A	7.02	58.1	326
	B	7.15	63.3	321
	C	6.55	56.1	279
	Mean	6.91	59.2	309
1.6	A	5.82	50.0	269
	B	5.90	55.1	291
	C	6.54	52.1	305
	Mean	6.09	52.4	288
3.1	A	6.08	48.9	299
	B	6.83	56.1	298
	C	7.20	57.5	316
	Mean	6.70	54.2	304
6.3	A	6.46	54.0	286
	B	7.46	65.1	345
	C	7.35	57.1	335
	Mean	7.09	58.7	322
13	A	5.53	48.0	247
	B	7.14	64.6	350
	C	7.00	58.7	313
	Mean	6.56	67.1	303
25	A	5.28	43.6	271
	B	6.27	54.5	279
	C	6.29	53.4	298
	Mean	5.95	50.5	262
50	A	4.59	36.1	217
	B	5.28	46.6	289
	C	6.02	49.9	279
	Mean	5.30	44.2	262
100	A	3.30	27.3	191
	B	3.16	27.0	188
	C	2.88	27.4	185
	Mean	3.11	27.2	188
Inoculum (Day 0) cell density = 1.03 x 10^4cells/mL

 

Table 4: Mean areas under the growth curve

Nominal concentration of test material (mg/L)	Mean area under growth curve (0-72 hours)	Percentage of culture medium control
Culture medium control	202	-
0.78	218	108
1.6	200	99
3.1	210	104
6.3	224	111
13	213	105
25	195	97
50	178	88
100	122	60*
* Significant difference (P = 0.05) from the culture medium control

Table 5: Mean growth rates over the test period

Nominal concentration of test material (mg/L)	Mean growth rate (0-72 hours)	Percentage of culture medium control
Culture medium control	1.88	-
0.78	1.90	101
1.6	1.88	100
3.1	1.90	101
6.3	1.91	102
13	1.90	101
25	1.87	100
50	1.85	98
100	1.74	92*
* Significant difference (P = 0.05) from the culture medium control

Table 6: Test solution pH measurements

Nominal concentration of test material (mg/L)	pH
	0 Hours	72 Hours
		Replicate A	Replicate B
Culture medium control	7.40	9.94	9.37
0.78	7.42	9.64	10.15
1.6	7.42	10.25	10.01
3.1	7.45	9.76	9.88
6.3	7.41	10.00	9.53
13	7.45	10.08	9.78
25	7.43	9.51	10.13
50	7.39	9.58	9.63
100	7.37	9.03	9.54

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the EC50s for growth rate and biomass of the test material in algae could not be determined as they were above the maximum concentration tested in the study. The EC50 for both growth and biomass was therefore determined to be >100 mg/L. A significant reduction in the growth rate of the algae was noted in the highest concentration however this did not reach 50 % and is therefore not sufficient to trigger a classification. Under the conditions of the test, the NOEC in Selenastrum capricornutum has been determined to be 50 mg/L. Although there were deviations present in the performance of this study, ultimately, theses were not considered to affect the assessment of the test substance. The study was considered suitable for assessment of the classification and labelling and risk assessment of the substance. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The toxicity of the test material to aquatic plants was investigated in the algae Selenastrum capricornutum in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850.5400. The algal culture was exposed to the test material in at nominal concentrations of 0.78, 1.6, 3.1, 6.3, 13, 25, 50 and 100 mg/L under static conditions. The mean measured concentrations of the test substance were ranged from 92 to 106 % of the nominal exposure concentrations; the results and conclusions were therefore based on the nominal concentrations. The EC₅₀s for growth rate and biomass of the test material in algae could not be determined as they were above the maximum concentration tested in the study. The EC₅₀ for both growth and biomass is therefore considered to be >100 mg/L under the conditions of the test. A significant reduction in the growth rate of the algae was noted in the highest concentration however this did not reach 50 % and is therefore not sufficient to trigger a classification. Under the conditions of the test, the NOEC in Selenastrum capricornutum has been determined to be 50 mg/L.

Description of key information

72 h EC50(growth rate) >100 mg/L, NOEC = 50 mg/L, Selenastrum capricornutum, OECD 201, Maynard 2003

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

The toxicity of the test material to aquatic plants was investigated in the algae Selenastrum capricornutum in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850.5400. The algal culture was exposed to the test material in at nominal concentrations of 0.78, 1.6, 3.1, 6.3, 13, 25, 50 and 100 mg/L under static conditions. The mean measured concentrations of the test material were ranged from 92 to 106 % of the nominal exposure concentrations; the results and conclusions were therefore based on the nominal concentrations. The EC50s for growth rate and biomass of the test material in algae could not be determined as they were above the maximum concentration tested in the study. The EC50 for both growth and biomass is therefore considered to be >100 mg/L under the conditions of the test. A significant reduction in the growth rate of the algae was noted in the highest concentration however this did not reach 50 %. Under the conditions of the test, the NOEC in Selenastrum capricornutum has been determined to be 50 mg/L. The study was performed in line with GLP and accepted standardised guidelines with a high standard of reporting. However, minor deviations to the standardised guideline of the study were found to be present and as such the study was assigned a reliability score of 2 as reliable with acceptable restrictions. These deviations included a drift of greater than 1.5 pH units. Microscopic evaluations were not available to determine the effect of the pH on the integrity and morphology of the cells, however using the growth rates it was possible to demonstrate a lack of effect by the pH increase. The results were considered sufficient for risk assessment and classification and labelling purposes. The available data is considered to be complete and the result determined, 72 h NOEC value 50 mg/L, was taken forward for risk assessment.