Perhydro-1,3,5-trinitro-1,3,5-triazine

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:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental study performed according to the OECD 201 guideline and under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Please, see TMI

Analytical monitoring:

yes

Details on sampling:

The analytical samples were sampled at the beginning of the test in the test solutions preparations and at the end of the test in the content of the vessels pooled by replicate.
The following protocol was applied:
- Minimum of 1 mL of sample was collected and pipetted into a 2 mL micro-centrifugation tube;
- For control sample, LS/16, LS8, LS4 samples, 1 mL was collected with a syringe and was filtered (Filter Macherey Nagel RC-45/13 ref 729237) into a 1.5 mL glass amber vial;
- For LS/2 and LS samples, 100 µL was diluted with 900 µL of algae medium before filtration of 1 mL.

Details on test solutions:

Test item was supplied by the sponsor as a saturated solution. Aqueous phase of this solution was used as Limit of Solubility solution (LS). As this saturated solution was prepared with deionised water, stock solutions of media were added at LS solution as below:
Test item solution (LS) = 500 mL
Media stock solution (SM1) = 5 mL
Media stock solution (SM2) = 0.5 mL
Media stock solution (SM3) = 0.5 mL
Media stock solution (SM4) = 0.5 mL
Final volume (F) = 506.5 mL
Dilution factor (F / LS) = 1.013 mL
Dilution factor (1.013) was negligible to performed the test at LS concentration.
The test solution was then prepared by dilution of test item solution

Test organisms (species):

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

Details on test organisms:

The Pseudokirchneriella subcapitata stock culture was maintained at 4°C and regularly transferred to fresh medium at the laboratory. The algal stock culture was provided by the CCAP (Culture Collection of Algae and Protozoa), reference CCAP 278/4. A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers to fresh medium used in the test (OECD medium) every 3 to 4 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Hardness:

Not specified

Test temperature:

22.4 to 23.0 °C (mean: 22.7°C)

pH:

7.6 - 8.0

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0; LS/16; LS/8; LS/4; LS/2 and LS
Measured (T0): 0; 2.03, 3.98, 8.00, 16.37 and 32.36 mg/L
Measured (72h): 0; 2.01, 3.99, 7.99, 16.13 and 32.28 mg/L

Details on test conditions:

Based on the results of the no-GLP range-finding test, the test solutions were prepared in OECD medium at the definitive nominal concentrations of LS/16; LS/8; LS/4; LS/2 and LS. A control was performed at the same time with test media only.
Every flask was inoculated with the initial cell concentration of 5*10^3 cells.mL-1 of Pseudokirchneriella subcapitata as recommended in the guideline OECD 201.

Starting from a culture of 3.55E+06 cells per millilitres, 705 µL were inoculated in 500 mL of OECD medium for the control (C02) and 352.5 µL were inoculated in 250 mL of OECD medium for the tested concentrations (C03 to C07), to reach a concentration of about 5000 cells per milliliters. The preparations were divided in 6 replicates for the control and 3 replicates for the tested concentrations consisting in about 80 mL per replicates.
The flasks consisted of glass Erlenmeyer flasks of 250 mL capacity, closed with air-permeable stoppers made of polyether foam.
The test was performed in a thermostatic chamber maintained in the range of 21°C to 24°C controlled to ± 2°C under continuous illumination (with a light intensity between 4440 and 8880 lux). Algae were maintained suspended under continuous orbital stirring.
According to the study plan, cell biomass was measured every day in each flask using an electronic particle counter.
At the end of the test, after 72 hours of exposure, a microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.
pH was recorded at the beginning and the end of the test in all treatments.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 32.36 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

EC50 was above the solubility limit of the substance and could therefore not be determined

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32.36 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Corresponding to the water solubility limit of the substance

Details on results:

See the section "any information on results incl. tables".
At the end of the test, after 72 hours of exposure, a microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

Results with reference substance (positive control):

Potassium dichromate: EC-72h-50 (mg.L-1): 1.09
95% confidence limits (mg.L-1): 0.95 – 1.23

Table 1: Cells number on control flasks

	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)
Control C02	5000	29608	206600	1474000
	5000	27024	209100	1146000
	5000	23256	172300	1211000
	5000	18792	170100	1073000
	5000	14384	159700	1277000
	5000	29384	139000	1104000
Mean	5000	23741	176133	1214167

 

Table 2: Specific growth rates, growth rates and yields of control replicates

	Specific growth rate 0 h – 24 h	Specific growth rate 24 h – 48 h	Specific growth rate 48 h – 72 h	Mean of specific rate per replicate	Standard deviation of specific rate	CV of specific rate	Growth rate (day^-1) after 72h	CV growth 72h (%)	Yield (cells / mL) after 72h	CV yield 72h (%)
Control C02	1.779	1.943	1.965	1.895	0.102	5.370	1.895	2.1	1469000	12.2
	1.687	2.046	1.701	1.812	0.203	11.220	1.812		1141000
	1.537	2.003	1.950	1.830	0.255	13.931	1.830		1206000
	1.324	2.203	1.842	1.790	0.442	24.687	1.790		1068000
	1.057	2.407	2.079	1.848	0.704	38.122	1.848		1272000
	1.771	1.554	2.072	1.799	0.260	14.465	1.799		1099000
Mean	1.526	2.026	1.935	1.829	0.328	17.966	1.829		1209167

 

Table 3: Cells number, growth rates and yields in the different replicates of the test item group

Rates	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)	Growth rate (day^-1) after 72h	CV growth 72h (%)	Growth rate inhibition (%) compared to control after 72h	Yield (cells / mL) after 72h	CV yield 72h (%)	Yield inhibition (%) compared to control after 72h
mg test item/L after 72h
LS/16 (C03)	5000	24336	144200	1139000	1.809	3.4	1.06	1134000	19.7	6.22
	5000	31776	191900	1578000	1.918		-4.88	1573000		-30.09
	5000	14016	140500	1144000	1.811		0.98	1139000		5.80
Mean	5000	23376	158867	1287000	1.846		-0.95 (ns)	1282000		-6.02 (ns)
LS/8 (C04)	5000	29392	158300	1025000	1.774	2.8	2.98	1020000	16.0	15.64
	5000	29504	179000	1335000	1.862		-1.83	1330000		-9.99
	5000	29016	181600	1024000	1.774		3.00	1019000		15.73
Mean	5000	29304	172967	1128000	1.804		1.38 (ns)	1123000		7.13 (ns)
LS/4 (C05)	5000	18728	153400	1065000	1.787	1.9	2.28	1060000	10.4	12.34
	5000	29456	182200	1289000	1.851		-1.20	1284000		-6.19
	5000	32928	158000	1106000	1.800		1.60	1101000		8.95
Mean	5000	27037	164533	1153333	1.813		0.89 (ns)	1148333		5.03 (ns)
LS/2 (C06)	5000	31240	151800	560400	1.573	3.3	13.99	555400	16.0	54.07
	5000	20328	148900	769900	1.679		8.20	764900		36.74
	5000	13192	123300	651900	1.623		11.23	646900		46.50
Mean	5000	21587	141333	660733	1.625		11.14 (ns)	655733		45.77 (ns)
LS (C07)	5000	27952	146000	642600	1.619	1.0	11.49	637600	5.1	47.27
	5000	29928	152400	704700	1.649		9.81	699700		42.13
	5000	21640	128300	652200	1.624		11.22	647200		46.48
Mean	5000	26507	142233	666500	1.631		10.84 (ns)	661500		45.29 (ns)

ns: not statisticallysignificant compared to the control (p>0.05, Dunn test)

* statistically significant compared to the control (p<0.05, Dunn test)

 

Table 4: pH measurements

	C02	C03	C04	C05	C06	C07
pH at 0 h	7.7	7.7	7.7	7.7	7.7	8.0
pH at 72 h	7.7	7.6	7.6	7.6	7.6	7.9

 

Table 5: Temperature and light intensity during the test

Time	Mean	Min	Max
Temperature (TMP_0073)	22.7	22.4	23.0
Light intensity (lux)	8085

Table 6: Validity criteria

	Actual value	Reference value		Compliance
Growth multiplication factor in the control within the 72-hour test period	243	>	16	Y
Coefficient of variation of the average specific growth rates in the control during the whole test period	2.12	<	7	Y
Mean coefficient of variation for section-by-section specific growth rates (days 0-1. 1-2 and 2-3) in the control	17.97	<	35	Y

Table 7: Analytical results

ID	Concentrations	0h(mg.L^-1)	72h(mg.L^-1)
C02	0	0	0
C03	LS/16	2.03	2.01
C04	LS/8	3.98	3.99
C05	LS/4	8.00	7.99
C06	LS/2	16.37	16.13
C07	LS	32.36	32.28

Validity criteria fulfilled:

yes

Conclusions:

RDX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. EC50 based on growth rate could not be determined because it was above the solubility limit of the substance, i.e. 32.36 mg/L.
The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 32.36 and 32.36 mg/L, respectively. The EC10 was 23.12 mg/L (95% confidence limits: 15.23 – 33.44 mg/L).

Executive summary:

The objective of this study was to assess the effects of test item Hexogen solution on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201.

The test item is a saturated solution supplied by the sponsor. The test solutions were prepared in the test medium by dilution of saturated test item solution, and were expressed in limit of solubility (LS). The effects of Hexogen solution at the definitive nominal concentrations of 0; LS/16; LS/8; LS/4; LS/2 and LS to Pseudokirchneriella subcapitata were investigated under laboratory conditions.

The analytical samples were sampled at the beginning of the test and at the end of the test (72 hours) with the content of the vessels pooled and homogenized.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

The EC10, EC20, EC50 and NOEC/LOEC values are summarized in the table below:

	Hexogen solution
NOEC (growth rate and yield)	32.36 mg.L^-1
LOEC (growth rate and yield)	>32.36 mg.L^-1
72h-ErC10 (95% confidence limits)	23.12 mg.L^-1(95% confidence limits: 15.23 – 33.44 mg.L^-1)
72h-ErC20 (95% confidence limits)	>32.36 mg.L^-1
72h-ErC50 (95% confidence limits)	>32.36 mg.L^-1
72h-EyC10 (95% confidence limits)	5.65 mg.L^-1(95% confidence limits: 1.76 – 14.44 mg.L^-1)
72h-EyC20 (95% confidence limits)	10.72 mg.L^-1(95% confidence limits: 5.27 – 20.07 mg.L^-1)
72h-EyC50 (95% confidence limits)	31.98 mg.L^-1(95% confidence limits: 22.28 – 49.57 mg.L^-1)

EC = Effect concentration

NOEC= No Observed Effect Concentration

LOEC = Lowest Observed Effect Concentration

Description of key information

For that endpoint, an experimental study was available to assess the effects of test item on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline and under GLP conditions.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

RDX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. EC50 based on growth rate was above the solubility limit of the substance, i.e. 32.36 mg/L.

The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 32.36 and 32.36 mg/L, respectively. The EC10 was 23.12 mg/L (95% confidence limits: 15.23 – 33.44 mg/L).

All validity criteria were met, therefore this study was considered valid.

Additional information

For that endpoint, quite similar results were found in the study from Burton et al. (publication, 1994) performed on RDX according to EPA TSCA Method 797.1060 (U.S. EPA, 1985 and 1986).

In this study, a maximum reduction of 38% in cell density occured after a 96-h exposure to the solubility limit of the substance, i.e. 36.7 mg/L ; thus, an EC50 could not be determined.

The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) for the green alga Selenastrum capricornutum were 4.8 and 0.5 mg/L, respectively, when chronic endpoints were used to analyze the data. Nevertheless, the key parameter investigated was the inhibition of growth after 96 hours (i.e. 96h-EC50) based on yield rather than growth rate (logarithmic increase in biomass). Therefore this study does not provide an adequate coverage of the key parameter foreseen to be investigated. In addition, the mean coefficient of variation for section-by-section specific growth rates in the control cultures was above 35% meaning that the validity criteria were not all successfully met when compared with those of the test methods referred to in Article 13(3), i.e. EU C.3 and OECD TG 201. For these reasons, this study has been finally disregarded.