1,3-Benzenedimethanamine, reaction products with polyethylene glycol mono-Bu ether and TDI

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(on Day 2 of the test, the light intensity exceeded the mean by more than 15% (16%) and during the first two days of incubation the incubator temperature raised to max. 25.5 °C. This deviation did not adversely impact the results of this study.)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(on Day 2 of the test, the light intensity exceeded the mean by more than 15% (16%) and during the first two days of incubation the incubator temperature raised to max. 25.5 °C. This deviation did not adversely impact the results of this study.)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the test, two samples (50 mL) were taken from the freshly-prepared uninoculated control and test media. After 72 hours, two samples (50 mL) of control and test media were taken from uninoculated replicate flasks. Samples were stored in a freezer prior to analysis.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
9.54 mg/10 litres, 15.25 mg/5 litres,9.77 mg/litre, 31.3 mg/1 litre and 100 mg/1 litre was dispersed in dilution medium (algal nutrient medium as recommended in the guidelines) in glass vessels. The contents of each vessel were stirred for approximately 24 hours and then left to stand for approximately 24 hours. Aliquots (800 mL) were then removed mid vessel to provide the Water Accomodated Fractions (WAF).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth.

ACCLIMATION
- Acclimation period: 6 days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

25 mg/L as CaCO3

Test temperature:

21.4-25.5 °C
During the first two days of incubation the incubator temperature raised to max. 25.5 °C. At test end, the temperature was 24 °C. As the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.

pH:

test start (0 h): 7.6-7.7
test end (72 h): 8.3-8.7

Nominal and measured concentrations:

nominal loading rates: 0.954, 3.05, 9.77, 31.3 and 100 mg/L as WAF
measured TOC levels:
- loading rates of 0.954, 3.05 and 9.77 mg/L: below the limit of quantification (after background correction)
- loading rates of 31.3 and 100 mg: 0.282 and 2.18 mg C/L at test start and 0.0837 and 1.40 mg C/L at test end;
overall mean measured TOC levels: 0.154 and 1.75 mg C/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 118 x10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 1.0 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 6148 to 6710 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.
On Day 2, the light intensity exceeded the mean by 16%. As the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth after 72 hours was 21% at 10 mg/L and 29% at 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

45.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits: 11-95 mg/L; results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

55.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 20-95 mg/L; results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rates

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: minor stimulation was observed (see Table 2). This had no influemce on calculation of EC50 values.

At the start of the test, the test media (as WAF) at all nominal loading rates were colourless dispersions.

Results with reference substance (positive control):

- 72-hour EbC50: 0.541 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)

Reported statistics and error estimates:

Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates. 95% confidence intervals were calculated using the likelihood ratio method. Williams’ test was used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test was used.

Table 1: Cell densities

 

Nominal loading rates (mg/L)	Replicate number	Cell densities (cells/mL)
		24 hours	48 hours	72 hours
Control	R1	53450	243283	1111300
	R2	50717	251717	1109933
	R3	48217	249250	1196100
	R4	48350	242450	1115600
	R5	49983	259017	1301333
	R6	58117	297350	1253033
	Mean	51472	257178	1181217
0.954	R1	49517	262483	991400
	R2	48783	266350	1007267
	R3	54917	255350	1415433
	Mean	51072	261394	1138033
3.05	R1	54950	265950	1027733
	R2	58817	291683	1512400
	R3	64517	297650	1438600
	Mean	59428	285094	1326244
9.77	R1	54950	259950	1257233
	R2	54183	273483	1225033
	R3	52983	262450	1254367
	Mean	54039	265294	1245544
31.3	R1	55683	282050	1252333
	R2	50783	247717	929967
	R3	49750	301417	1224833
	Mean	52072	277061	1135711
100	R1	42383	194783	848167
	R2	42283	178650	731600
	R3	46150	204550	870933
	Mean	43606	192661	816900

 

R1-R6: replicate number

Note: the initial cell density was estimated to be 0.865 x 10⁴/mL

 

 

Table 2  Inhibition of growth

 

Parameter	Nominal loading rates (mg/L)	Sample size	Mean	Inhibition (%)	p-value
Area under curve to 72 hours	Control	6	21.0	0.0	-
	0.0954	3	20.6	2.0	>0.999^W
	0.305	3	23.6	-12.4	>0.999^W
	0.977	3	22.0	-4.9	>0.999^W
	3.13	3	20.9	0.3	>0.999^W
	10	3	14.9	29.1	0.001**^W
Growth rate to 72 hours	Control	6	0.066	0.0	-
	0.0954	3	0.066	1.0	>0.999^W
	0.305	3	0.068	-2.2	>0.999^W
	0.977	3	0.067	-1.2	>0.999^W
	3.13	3	0.066	1.0	0.832^W
	10	3	0.061	7.8	0.001**^W
Growth rate 0 to 24 hours	Control	6	0.068	0.0	-
	0.0954	3	0.068	0.4	>0.999^D
	0.305	3	0.074	-8.8	0.025*^D
	0.977	3	0.070	-3.1	0.731^D
	3.13	3	0.069	-0.8	>0.999^D
	10	3	0.061	10.1	0.010**^D
Growth rate 24 to 48 hours	Control	6	0.067	0.0	-
	0.0954	3	0.068	-1.6	>0.999^W
	0.305	3	0.065	2.4	>0.999^W
	0.977	3	0.066	1.1	>0.999^W
	3.13	3	0.070	-3.8	>0.999^W
	10	3	0.062	7.7	0.018*^W
Growth rate 48 to 72 hours	Control	6	0.064	0.0	-
	0.0954	3	0.061	4.4	>0.999^W
	0.305	3	0.064	0.0	>0.999^W
	0.977	3	0.064	-1.4	>0.999^W
	3.13	3	0.059	7.9	0.289^W
	10	3	0.060	5.4	0.289^W
Yield	Control	6	1171217	0.0	-
	0.0954	3	1128033	3.7	>0.999^W
	0.305	3	1316244	-12.4	>0.999^W
	0.977	3	1235544	-5.5	>0.999^W
	3.13	3	1125711	3.9	0.899^W
	10	3	806900	31.1	0.005**^W

 

p values are for the comparison with Control using Williams' test (W) and Dunnett's test (D)

*p< 0.05, **p< 0.01

 

 

Control cultures:

-cell concentration increased by a factor of 118;

-the mean coefficient of variation for daily growth rates ranged between 3.0 and 4.4%;

- the coefficient of variation for the average specific growth rates was 1.5% during the 72 hour exposure period.

Validity criteria fulfilled:

yes

Remarks:

The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD TG 201, EU C.3):
EC50: >100 mg/L, NOELR: 31.3 mg/L, EC10: >100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

31.3 mg/L

Additional information

The EC50/LC50 for the test material is much higher than its water solubility.