In a bad ending in Jade's route in Volume 4, the MSPA Reader attacks Jade's dog Becquerel. Although he is not killed, the description of physically attacking a dog may upset people.
In Eridan's route in Volume 10, the MSPA reader is said to puke all over the floor at Eridan's classist attitude. However, no visuals of them puking are shown, it is just stated in the narrative.
Unsure if it counts since it's the whole arm, but in a bad ending in Eridan's route in Volume 10, the MSPA Reader's arm gets blown off by Eridan's gun.
In Volume 14 in Dirk's route, very brief background images appear of the MSPA Reader slicing their fingers off with Dirk's katana, although this is presented as a hypothetical.
In Vriska's route, it is implied that she is a trans woman. One of these ways that this is reinforced is by her saying that her lusus doesn't call her by her chosen name.
The most prominent example of this is in Eridan's route in Volume 10. Eridan is genocidal in his attitudes towards lowbloods - who are seen to be the less privileged members of troll society. He says that lowbloods deserve to be culled just for having less satisfactory blood in their veins.
In a bad ending in Eridan's route in volume 10, the MSPA Reader gets their arm blown off by Eridan's gun. In Vriska's route in Volume 6, she is shown to have a prosthetic arm, but the amputation itself is only shown in Homestuck proper.
In Feferi's route in Volume 10, the mspa reader almost drowns, but manages to escape regardless of the player's choice at the time. In Dirk's route in Volume 14, brief background images of the MSPA reader drowning in Dirk's scuba gear appear, although this is presented as a hypothetical possibility.
Although they aren't outright stated as anxiety attacks, they appear similar.
In Volume 9, Nepeta is described to be trembling when she is zapped back to Alternia with Dave and Jade; also in Volume 9, Sollux becomes visibly emotionally distressed when another troll tells him to shut up on a train, which the player can choose to calm down.
There is a single sprite of one of the characters spitting out alcohol after trying it for the first time, but there are no jokes about spitting or farting
The MSPA reader has fourth-wall-breaking powers throughout - in the epilogue they meet the director of the game and Ultimate Dirk, who also break the fourth wall.
In Eridan's route in Volume 10, Sollux mentions that Eridan once asked him to install a wiretap into Feferi's hive so that Eridan could listen in on her - although Sollux refused to do this, it still counts as a stalkerish attitude.
In Volume 3, Dave mentions that there is never enough food to eat at home, and that he is being recorded by his Bro for an online audience. Readers of Homestuck will also know that Dave is forced to fight his Bro regularly.
In Volume 6, Gamzee speaks about his absentee lusus and how he disapproves of Gamzee's lifestyle.
Also in Volume 6, Vriska tells the reader that her spider lusus forces her to feed the lusus trolls that she captures, which has a negative mental effect on Vriska.
In Volume 6, Gamzee's route features sopor slime pie - consuming the slime has a hallucinogenic effect on the MSPA Reader in the same way as regular drugs. In the same route, there is also the implication that Gamzee snorts cocaine. In Volume 7 in Terezi's route, a bad ending features Terezi and the main character going to Gamzee's hive to use some more drugs.
In Volume 2, it is stated that Mom Lalonde is an alcoholic.
In Volume 6, it is shown that Gamzee is addicted to sopor slime, and that denying him his slime makes Gamzee go on a murderous rampage.
Rose's mom is said to be an alcoholic in Volume 2. A scene revolves around the MSPA Reader stealing a large amount of alcohol from Mom Lalonde's liquor cabinet.
Also in Jane's route in Volume 11, it is stated that Roxy drinks alcohol, despite being underaged at the time.
In a bad ending of Nepeta's route in Volume 9, Jade shoots Nepeta's cat lusus Pounce De Leon in self defence, and it's heavily implied if not outright confirmed that Pounce is killed.
In the first bad ending of Terezi's route in Volume 7, the MSPA reader and Terezi fall out of Terezi's treehouse window, only for Terezi's neck to get caught in a noose. She is accidentally hanged and killed.
In one of the endings for Volume 3, the main character is thrown out of the window of a high-rise apartment building. The actual death is not shown. The ending-card is a cartoony image of the main character deep in thought about another subject entirely, as they plummet to the city below.
In Volume 7 Terezi, who is 13 at the time, is accidentally hanged and killed. In Volume 6 in one of Gamzee's bad endings, it is implied that Karkat and Kanaya, who are also 13 at the time, have been killed.
Although not "biological" parents, lusii are guardians. Vriska's spider lusus is killed in Vriska's good ending in Volume 6, and Nepeta's cat lusus is killed in a bad ending in Volume 9.
Volume 6 features Gamzee Makara, a clown or juggalo-like troll. This route also features Karako Pierot, another clownlike troll.
Gamzee also appears in Terezi's route in Volume 7, Sollux's route in Volume 9, and Jane's route in Volume 11.
In Jane's route in Volume 11, in one of the bad endings the MSPA reader puts on Jane's Unreal Heiress Thoughtwave Tiaratop and is brainwashed into serving the Condesce.
In Volume 8, it is mentioned that Vriska calls Tavros useless for being unable to climb stairs or fight, despite knowing about and being the cause of Tavros being disabled.
In one of Equius' bad endings in Volume 7, the reader witnesses several hypothetical flashbacks while trying to work out how Equius broke one of his horns. One of these flashbacks features Equius being hit by a scuttlebuggy - a troll car.